Genetic differences between blight-causing Erwinia species with differing host specificities, identified by suppression subtractive hybridization

PCR-based subtractive hybridization was used to isolate sequences from Erwinia amylovora strain Ea110, which is pathogenic on apples and pears, that were not present in three closely related strains with differing host specificities: E. amylovora MR1, which is pathogenic only on Rubus spp.; Erwinia pyrifoliae Ep1/96, the causal agent of shoot blight of Asian pears; and Erwinia sp. strain Ejp556, the causal agent of bacterial shoot blight of pear in Japan. In total, six subtractive libraries were constructed and analyzed. Recovered sequences included type III secretion components, hypothetical membrane proteins, and ATP-binding proteins. In addition, we identified an Ea110-specific sequence with homology to a type III secretion apparatus component of the insect endosymbiont Sodalis glossinidius, as well as an Ep1/96-specific sequence with homology to the Yersinia pestis effector protein tyrosine phosphatase YopH.     

Genetic Differences between Blight-Causing Erwinia Species with Differing Host Specificities, Identified by Suppression Subtractive Hybridization

PCR-based subtractive hybridization was used to isolate sequences from Erwinia amylovora strain Ea110, which is pathogenic on apples and pears, that were not present in three closely related strains with differing host specificities: E. amylovora MR1, which is pathogenic only on Rubus spp.; Erwinia pyrifoliae Ep1/96, the causal agent of shoot blight of Asian pears; and Erwinia sp. strain Ejp556, the causal agent of bacterial shoot blight of pear in Japan. In total, six subtractive libraries were constructed and analyzed. Recovered sequences included type III secretion components, hypothetical membrane proteins, and ATP-binding proteins. In addition, we identified an Ea110-specific sequence with homology to a type III secretion apparatus component of the insect endosymbiont Sodalis glossinidius, as well as an Ep1/96-specific sequence with homology to the Yersinia pestis effector protein tyrosine phosphatase YopH.     

Genetic Transfer of Episomic Elements Among Erwinia Species and Other Enterobacteria: F′lac+

The episomic element F'lac(+) was transferred, probably by conjugation, from Escherichia coli to Lac(-) strains of Erwinia herbicola, Erwinia amylovora, and Erwinia chrysanthemi (but not to several other Erwinia spp. In preliminary trials). The lac genes in the exconjugants of the Erwinia spp. showed varying degrees of stability depending on the strain (stable in E. herbicola strains Y46 and Y74 and E. amylovora strain EA178, but markedly unstable in E. chrysanthemi strain EC16). The lac genes and the sex factor (F) were eliminated from the exconjugants by treatment with acridine orange, thus suggesting that both lac and F are not integrated in the Erwinia exconjugants. All of the tested Lac(+) exconjugants of E. herbicola strains Y46 and Y74 and E. amylovora strain EA178, but not of E. chrysanthemi strain EC 16, were sensitive to the F-specific phage M13. The heterogenotes (which harbored F'lac(+)) of E. herbicola strains Y46 and Y74, E. amylovora strain EA178, and E. chrysanthemi strain EC16 were able to transfer lac genes by conjugation to strains of E. herbicola, E. amylovora, E. chrysanthemi, Escherichia coli, and Shigella dysenteriae. The frequency of such transfer from Lac(+) exconjugants of Erwinia spp. was comparable to that achieved by using E. coli F'lac(+) as donors, thus indicating the stability, expression, and restriction-and-modification properties of the sex factor (F) in Erwinia spp.     

伤寒沙门菌非编码RNA617的分子鉴定及其对生物膜形成的影响研究

本研究旨在探讨伤寒沙门菌(Salmonella enterica serovar Typhi, S. Typhi)中非编码RNA617(non-coding RNA617,ncRNA617)的分子特性,并研究其对生物膜形成的影响及作用机制。采用Northern blot方法检测ncRNA617的表达,通过cDNA 5’末端快速扩增技术(5’-rapid amplification of cDNA end,5’RACE)和逆转录-聚合酶链式反应(reverse transcriotion-polymerase chain reaction,3’RT-PCR)实验分析ncRNA617可能的转录起始位点和终止位点;构建ncRNA617缺陷菌株、回补菌株和过表达菌株等相关菌株,通过生物膜形成实验,观察ncRNA617对伤寒沙门菌生物膜形成的影响,并用实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qPCR)分析生物膜形成相关基因表达水平的变化,综合运用生物信息学方法预测ncRNA617和差异基因的结合区域,初步分析ncRNA617发挥调控作用的机制。结果显示,伤寒沙门菌确有ncRNA617的表达,长度约300 nt,其转录起始位点位于mig-14终止密码子下游967 nt处,终止位点位于t2681起始密码子上游 2 378～2 560 nt处。与野生对照菌株相比,ncRNA617缺陷菌株生物膜形成能力增强(P<0.05),回补菌株的生物膜形成能力恢复至野生菌株水平,过表达菌株的生物膜形成能力有所下降(P<0.05)。qPCR结果表明,ncRNA617可负向调控多个生物膜形成相关基因的转录表达水平(P<0.05)。经生物信息学方法预测发现,ncRNA617与差异基因有不同的结合区域。本研究结果提示,ncRNA617在伤寒沙门菌中存在,其长度约270～452 nt。ncRNA617可能通过靶向结合生物膜形成相关基因下调基因表达,从而负向调控伤寒沙门菌生物膜的生成。     

Erwinia spp. from pome fruit trees: similarities and differences among pathogenic and non-pathogenic species

The number of described pathogenic and non-pathogenic Erwinia species associated with pome fruit trees, especially pear trees, has increased in recent years, but updated comparative information about their similarities and differences is scarce. The causal agent of the fire blight disease of rosaceous plants, Erwinia amylovora, is the most studied species of this genus. Recently described species that are pathogenic to pear trees include Erwinia pyrifoliae in Korea and Japan, Erwinia spp. in Japan, and Erwinia piriflorinigrans in Spain. E. pyrifoliae causes symptoms that are indistinguishable from those of fire blight in Asian pear trees, Erwinia spp. from Japan cause black lesions on several cultivars of pear trees, and E. piriflorinigrans causes necrosis of only pear blossoms. All these novel species share some phenotypic and genetic characteristics with E. amylovora. Non-pathogenic Erwinia species are Erwinia billingiae and Erwinia tasmaniensis that have also been described on pome fruits; however, less information is available on these species. We present an updated review on the phenotypic and molecular characteristics, habitat, pathogenicity, and epidemiology of E. amylovora, E. pyrifoliae, Erwinia spp. from Japan, E. piriflorinigrans, E. billingiae, and E. tasmaniensis. In addition, the interaction of these species with pome fruit trees is discussed.     

Nonsense-suppressor mutants of Erwinia carotovora subsp. carotovora

Abstract A promiscuous plasmid (pLM2) carrying amber mutations in two antibiotic-resistance genes was transferred to a derivative of Erwinia carotovora subsp. carotovora strain SCRI193. Following mutagenesis, two putative amber-suppressing mutants of this strain were isolated. The genotype of these mutants was confirmed by use of rep _am plasmid-specific phage. This constitutes the first isolation of amber-suppressing mutants in Erwinia spp.     

Mu-lac insertion-directed mutagenesis in a pectate lyase gene of Erwinia chrysanthemi.

The pelC gene, which encodes one of the five major pectate lyase (PL) isoenzymes in Erwinia chrysanthemi 3937, designated PLc, was subcloned from a hybrid lambda phage into a pBR322 derivative and mutagenized with a mini-Mu-lacZ transposable element able to form fusions to the lacZ gene. One plasmid (pAD1) which had an inactivated pelC gene and a Lac+ phenotype was selected in Escherichia coli. This plasmid was introduced into Erwinia chrysanthemi, and the pelC::mini-Mu insertion was substituted for the chromosomal allele by homologous recombination. This strain lacks the PLc isoenzyme. This Erwinia chrysanthemi strain has a Lac+ phenotype that is inducible by polygalacturonate, as are the wild-type PL activities.     

Transfer among Erwinia spp. and other enterobacteria of antibiotic resistance carried on R factors

Antibiotic resistance carried on R factors was transferred by conjugation from Escherichia coli B/r and Shigella flexneri 1a to Erwinia spp. Tetracycline resistance (TetR) carried on R factor R100 drd-56 was transferred from E. coli B/r to strains of Erwinia amylovora, E. aroideae, E. atroseptica, E. chrysanthemi, E. cytolytica, E. dissolvens, E. herbicola, E. nigrifluens, and E. nimipressuralis, but not to strains of Erwinia carotovora, E. carnegieana, E. dieffenbachiae, E. oleraceae, and E. quercina. Multiple antibiotic resistance (chloramphenicol, streptomycin, tetracycline; ChlR-StrR-TetR) carried on R factor SR1 was transferred from a clinical isolate of S. flexneri 1a to strains of E. aroideae, E. chrysanthemi, E. herbicola, and E. nigrifluens, but not to strains of other Erwinia spp. The frequency of this transfer was low with receptive cultures of Erwinia spp. and E. coli (F(-) strain). Antibiotic resistance in the exconjugants showed varying degrees of stability in the presence or absence of acridine orange, depending on the strain tested. The frequencies of segregation to drug susceptibility in the presence of acridine orange, though low, suggest that the elements exist as plasmids in the majority of the Erwinia exconjugants. Multiple antibiotic resistance (ChlR-StrR-TetR) was found to segregate into various resistance classes (ChlR-StrR, StrR-TetR, TetR, StrR, and none) in these exconjugants. The exconjugants of E. amylovora, E. herbicola, and E. nigrifluens, to which R100 drd-56 was transferred from E. coli B/r, were sensitive to the male (F)-specific phage M13. There was a positive correlation between the susceptibility of exconjugants to the F-specific phage M13 and their ability to transfer R100 drd-56 to the recipient cultures of Escherichia coli, Erwinia herbicola, Salmonella typhimurium, and Shigella dysenteriae. Exceptions were, however, noted with Erwinia dissolvens and E. nimipressuralis exconjugants harboring R100 drd-56; these exconjugants, although not susceptible to M13, transferred R100 drd-56 to the recipient cultures. The frequency of transfer of R100 drd-56 and the levels of resistance to tetracycline in Erwinia exconjugants were found to differ markedly depending upon the strain employed. Transfer of multiple antibiotic resistance (ChlR-StrR-TetR) from Erwinia exconjugants was not obtained in preliminary trials with an E. coli F(-) strain as the recipient culture.     

Taxonomic studies on Corynebacterium beticola (Abdou)

Morphological, physiological and chemical studies were performed on the type strain of Corynebacterium beticola in an attempt to clarify its taxonomy. The results indicate that Corynebacterium beticola is a Gram negative, facultatively anaerobic bacterium which should be assigned to the genus Erwinia as Erwinia herbicola .     

Molecular differentiation of Erwinia amylovora strains from North America and of two Asian pear pathogens by analyses of PFGE patterns and hrpN genes

In order to determine a possible genomic divergence of Erwinia amylovora'fruit tree' and raspberry strains from North America, several isolates were differentiated by pulsed-field gel electrophoresis (PFGE) analysis, the size of short DNA sequence repeats (SSRs) and the nucleotide and deduced amino acid sequences of their hrpN genes. By PFGE analysis European strains are highly related, whereas strains from North America were diverse and were further distinguished by the SSR numbers from plasmid pEA29. The E. amylovora strains from Europe showed identical HrpN sequences in contrast to the American isolates from fruit trees and raspberry. Those were related to each other, but distinguishable by their HrpN patterns. The Asian pear pathogens differed in HrpN among each other and from E. amylovora. Erwinia pyrifoliae isolates and the Erwinia strains from Japan were ordered via their HrpN sequences in agreement with the PFGE patterns. For all three pathogens, dendrograms from PFGE and sequence data indicate an evolutionary diversity within the species in spite of a genetic conservation for parts of the hrpN genes suggesting a long persistence of the Asian pear pathogens in Korea and Japan as well as of fire blight in North America. Some of the divergent American E. amylovora isolates share PFGE patterns with the relatively uniform European strains.     

Serological difference betweenErwinia herbicola strains of plant and human origin

FiftyErwinia herbicola isolates obtained from host plants were examined in an agglutination reaction with antiserum prepared againstE. ananas (E. herbicola) strain CCM 2407 antigen of plant origin and with antiserum prepared againstEnterobacter agglomerans strain CNCTC M 269 antigen of human origin. In tests with strain CCM 2407 antiserum, 56% isolates showed a positive reaction, while in tests with strain CNCTC M 269 antiserum only 14 % isolates showed a positive reaction. AmongE. herbicola isolates which showed a positive reaction with CCM 2407 antiserum 18 % showed a positive reaction with the CNCTC M 269 antiserum too. Our results confirmed the serological heterogeneity ofE. herbicola population. In spite of the difference in the origin of the two antigens used for the preparation of antisera (plant, human; Japan, Czech Republic) our results indicate that some of ourE. herbicola strains andE. agglomerans strain CNCTC M 269 are serologically identical.     

Cloning and characterization of a pectate lyase gene from Erwinia carotovora EC153

A pel gene cloned from strain EC153 of Erwinia carotovora encoded a pectate lyase that macerated plant tissue with moderate efficiency. This gene, called pel153, was sequenced and found to possess considerable homology with a pectate lyase gene from Yersinia pseudotuberculosis. The Yersinia protein, however, was truncated at the carboxyl terminal end relative to the Erwinia gene product and had a lower isoelectric point. The Erwinia pel153 gene was overexpressed in cells of Escherichia coli, and a 56-kDa protein was observed on sodium dodecyl sulfate-polyacrylamide gels. This compares with a molecular weight of 61 kDa for the mature, secreted protein as determined from sequencing data. Southern blot analysis disclosed the presence of the pel153 gene in three different strains of E. carotovora, but mutation of the gene in strain EC153 did not affect its ability to soft-rot potato tubers.     

Characterization of Erwinia amylovora strains using random amplified polymorphic DNA fragments (RAPDs)

The genetic diversity among 16 strains of Erwinia amylovora, chosen to represent different host plant origins and geographical regions, was investigated by RAPD analysis. One strain of Erwinia herbicola and one of Agrobacterium vitis were used as outgroups. Ninety-eight different RAPD fragments were produced by polymerase chain reaction amplification with six different 10-mer primers. RAPD banding profiles were found that enabled the Erw. amylovora strains to be distinguished from one another. Cluster analysis based on the number of RAPD fragments shared between strains showed that strains of Erw. amylovora isolated from subfamily Pomoideae formed a single group, whereas two strains from Rubus (subfamily Rosoideae) formed a second group. Two strains isolated from Asian pear on Hokkaido, Japan, formed a third group. Sets of RAPD fragments were identified that enabled each of the two host-range groups and one geographical region (Hokkaido) of Erw. amylovora strains to be unambiguously distinguished from one another and from the outgroups. This study shows that strains of Erw. amylovora exhibit genetic diversity detectable by RAPD analysis, and that molecular and statistical analysis of RAPD fragments can be used both to distinguish between strains and to determine relatedness between them.     

胡萝卜软腐欧文氏菌中信号分子合成酶expI基因的克隆、表达及其分析

用PCR方法从胡萝卜软腐欧文氏菌的基因组DNA中扩增出信号分子合成酶expI基因,将其克隆到大肠杆菌表达载体pET-28α(+)上,转化大肠杆菌BL21(DE3),获得高效表达expI基因的重组大肠杆菌BL21(pET28α-expI).重组菌经IPTG诱导表达,SDS-PAGE检测表达蛋白相对分子质量约为24.8kD,与预期分子量相符.经薄层层析和高效液相色谱分析发现该重组菌产生的信号分子种类为N-3-羰基己酰高丝氨酸内酯和N-己酰高丝氨酸内酯与胡萝卜软腐欧文氏菌产生的一致.     

The genome of Erwinia tasmaniensis strain Et1/99, a non-pathogenic bacterium in the genus Erwinia

The complete genome of the bacterium Erwinia tasmaniensis strain Et1/99 consisting of a 3.9 Mb circular chromosome and five plasmids was sequenced . Strain Et1/99 represents an epiphytic plant bacterium related to Erwinia amylovora and E. pyrifoliae , which are responsible for the important plant diseases fire blight and Asian pear shoot blight, respectively. Strain Et1/99 is a non-pathogenic bacterium and is thought to compete with these and other bacteria when occupying the same habitat during initial colonization. Genome analysis revealed tools for colonization, cellular communication and defence modulation, as well as genes coding for the synthesis of levan and a not detected capsular exopolysaccharide. Strain Et1/99 may secrete indole-3-acetic acid to increase availability of nutrients provided on plant surfaces. These nutrients are subsequently accessed and metabolized. Secretion systems include the hypersensitive response type III pathway present in many pathogens. Differences or missing parts within the virulence-related factors distinguish strain Et1/99 from pathogens such as Pectobacterium atrosepticum and the related Erwinia spp. Strain Et1/99 completely lacks the sorbitol operon, which may also affect its inability to invade fire blight host plants. Erwinia amylovora in contrast depends for virulence on utilization of sorbitol, the dominant carbohydrate in rosaceous plants. The presence of other virulence-associated factors in strain Et1/99 indicates the ancestral genomic background of many plant-associated bacteria.     

Type III secretion contributes to the pathogenesis of the soft-rot pathogen Erwinia carotovora: partial characterization of the hrp gene cluster

The virulence of soft-rot Erwinia species is dependent mainly upon secreted enzymes such as pectinases, pectin lyases, and proteases that cause maceration of plant tissue. Some soft-rot Erwinia spp. also harbor genes homologous to the hypersensitive reaction and pathogenesis (hrp) gene cluster, encoding components of the type III secretion system. The hrp genes are essential virulence determinants for numerous nonmacerating gram-negative plant pathogens but their role in the virulence of soft-rot Erwinia spp. is not clear. We isolated and characterized 11 hrp genes of Erwinia carotovora subsp. carotovora. Three putative sigmaL-dependent Hrp box promoter sequences were found. The genes were expressed when the bacteria were grown in Hrp-inducing medium. The operon structure of the hrp genes was determined by mRNA hybridization, and the results were in accordance with the location of the Hrp boxes. An E. carotovora strain with mutated hrcC, an essential hrp gene, was constructed. The hrcC- strain was able to multiply and cause disease in Arabidopsis, but the population kinetics were altered so that growth was delayed during the early stages of infection.     

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.     

Erwinia aphidicola,a new species isolated from pea aphid,Acyrthosiphon pisum

Five strains of Gram-negative, oxidase-negative, facultatively anaerobic, fermentative, motile, rod-shaped bacterium with the general characteristics of the family Enterobacteriaceae were isolated from the gut of multiple specimens of the pea aphid. All the strains caused aphid mortality when ingested by insects via a synthetic diet. The results of biochemical tests showed that these strains are most related to Erwinia herbicola and Pantoea agglomerans. According to DNA-DNA hybridization, the five strains showed more than 96% relatedness to each other, indicating that these organisms are members of a single species. These strains were most closely related to Erwinia herbicola (22% DNA relatedness). Phenotypic differentiation of these strains from Erwinia herbicola, which was also detected from aphid gut, was based on negative reactions in tests of yellow pigment production, gelatin liquefaction, acid production from inulin, starch and dulcitol, and positive acid production from melibiose, inositol, cellobiose and glycerol. On the basis of these data, the name Erwinia aphidicola is proposed for the new organism. The type strain is strain X 001 (=IAM 14479).