Erwinia herbicola isolates from alfalfa plants may play a role in nodulation of alfalfa by Rhizobium meliloti.

Erwinia herbicola was isolated from roots of plants derived from surface-sterilized seeds of all alfalfa varieties that were tested. Some of these E. herbicola strains affected nodulation by certain strains of Rhizobium meliloti. In previously published work we presented the isolation of slow-and fast-nodulating variants from a single culture of R. meliloti 102F51. In the absence of E. herbicola, the slow-nodulating variant induced the formation of nodules on alfalfa as rapidly as the faster-nodulating strain. The rates of nodulation by the faster-nodulating variant were the same in the presence and absence of E. herbicola. All of the previously reported slower-nodulating strains derived from R. meliloti 102F51 nodulated more rapidly on sterilized plants than in the presence of certain E. herbicola isolates.     

Erwinia herbicola isolates from alfalfa plants may play a role in nodulation of alfalfa by Rhizobium meliloti

Erwinia herbicola was isolated from roots of plants derived from surface-sterilized seeds of all alfalfa varieties that were tested. Some of these E. herbicola strains affected nodulation by certain strains of Rhizobium meliloti. In previously published work we presented the isolation of slow-and fast-nodulating variants from a single culture of R. meliloti 102F51. In the absence of E. herbicola, the slow-nodulating variant induced the formation of nodules on alfalfa as rapidly as the faster-nodulating strain. The rates of nodulation by the faster-nodulating variant were the same in the presence and absence of E. herbicola. All of the previously reported slower-nodulating strains derived from R. meliloti 102F51 nodulated more rapidly on sterilized plants than in the presence of certain E. herbicola isolates.     

草生欧文氏菌(E.herbicola)CSH1065质粒的重组标记及Fib基因的遗传转移

利用DNA分子克隆技术及遗传重组方法,在E.herbicola CSH1065质粒上插入了Km_R及Mob基因,利用细菌间的接合转移,使E.herbicola CSH1065质粒转移到E.coli HB101中,从而获得了E.herbicola CSH1065的Fib(fungi inhibition)基因在E.coli HB101中的表达,进一步证明E.herbicola CSH1065 Fib基因功能只涉及其细胞内的大质粒,与其染色体基因组无关,其黄色色素基因与Fib功能无关。这些结果还为DNA分子杂交方法所证实。     

Cloning and regulation of Erwinia herbicola pigment genes.

The genes coding for yellow pigment production in Erwinia herbicola Eho10 (ATCC 39368) were cloned and localized to a 12.4-kilobase (kb) chromosomal fragment. A 2.3-kb AvaI deletion in the cloned fragment resulted in the production of a pink-yellow pigment, a possible precursor of the yellow pigment. Production of yellow pigment in both E. herbicola Eho10 and pigmented Escherichia coli clones was inhibited by glucose. When the pigment genes were transformed into a cya (adenylate cyclase) E. coli mutant, no expression was observed unless exogenous cyclic AMP was provided, which suggests that cyclic AMP is involved in the regulation of pigment gene expression. In E. coli minicells, the 12.4-kb fragment specified the synthesis of at least seven polypeptides. The 2.3-kb AvaI deletion resulted in the loss of a 37K polypeptide and the appearance of a polypeptide of 40 kilodaltons (40K polypeptide). The synthesis of the 37K polypeptide, which appears to be required for yellow pigment production, was not repressed by the presence of glucose in the culture medium, as was the synthesis of other polypeptides specified by the 12.4-kb fragment, suggesting that there are at least two types of gene regulation involved in yellow pigment synthesis. DNA hybridization studies indicated that different yellow pigment genes exist among different E. herbicola strains. None of six pigmented plant pathogenic bacteria examined, Agrobacterium tumefaciens C58, Cornyebacterium flaccumfaciens 1D2, Erwinia rubrifaciens 6D364, Pseudomonas syringae ATCC 19310, Xanthomonas campestris 25D11, and "Xanthomonas oryzae" 17D54, exhibited homology with the cloned pigment genes.     

Divergent allosteric patterns verify the regulatory paradigm for aspartate transcarbamylase

The native Escherichia coli aspartate transcarbamoylase (ATCase, E.C. 2.1.3.2) provides a classic allosteric model for the feedback inhibition of a biosynthetic pathway by its end products. Both E. coli and Erwinia herbicola possess ATCase holoenzymes which are dodecameric (2(c3):3(r2)) with 311 amino acid residues per catalytic monomer and 153 and 154 amino acid residues per regulatory (r) monomer, respectively. While the quaternary structures of the two enzymes are identical, the primary amino acid sequences have diverged by 14 % in the catalytic polypeptide and 20 % in the regulatory polypeptide. The amino acids proposed to be directly involved in the active site and nucleotide binding site are strictly conserved between the two enzymes; nonetheless, the two enzymes differ in their catalytic and regulatory characteristics. The E. coli enzyme has sigmoidal substrate binding with activation by ATP, and inhibition by CTP, while the E. herbicola enzyme has apparent first order kinetics at low substrate concentrations in the absence of allosteric ligands, no ATP activation and only slight CTP inhibition. In an apparently important and highly conserved characteristic, CTP and UTP impose strong synergistic inhibition on both enzymes. The co-operative binding of aspartate in the E. coli enzyme is correlated with a T-to-R conformational transition which appears to be greatly reduced in the E. herbicola enzyme, although the addition of inhibitory heterotropic ligands (CTP or CTP+UTP) re-establishes co-operative saturation kinetics. Hybrid holoenzymes assembled in vivo with catalytic subunits from E. herbicola and regulatory subunits from E. coli mimick the allosteric response of the native E. coli holoenzyme and exhibit ATP activation. The reverse hybrid, regulatory subunits from E. herbicola and catalytic subunits from E. coli, exhibited no response to ATP. The conserved structure and diverged functional characteristics of the E. herbicola enzyme provides an opportunity for a new evaluation of the common paradigm involving allosteric control of ATCase.     

CHORIZOCOCCUS HERBICOLA (MASKELL) (COMB. N): (HOMOPTERA: PSEUDOCOCCIDAE) A GRASS-INFESTING SPECIES IN AUSTRALIA

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The Maskell species Dactylopius herbicola , a mealybug infesting grass in Australia, is redescribed and designated as a new combination, Chorizococcus herbicola (Maskell).     

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.     

Evaluation of two surface sampling methods for detection of Erwinia herbicola on a variety of materials by culture and quantitative PCR

This research was designed to evaluate surface sampling protocols for use with culture and quantitative PCR (QPCR) amplification assay for detection of the gram-negative bacterial biothreat simulant Erwinia herbicola on a variety of surface materials. Surfaces selected for evaluation were wood laminate, glass and computer monitor screens, metal file cabinets, plastic arena seats, nylon seat cushions, finished concrete flooring, and vinyl tile flooring. Laboratory and test chamber studies were performed to evaluate two sampling methods, a sponge and a macrofoam swab, for detection of E. herbicola on surface materials. In laboratory trials, seven materials were inoculated with a known concentration of E. herbicola cells and samples were collected from the surfaces of the materials to determine sampling efficiencies. Culture analysis was ineffective for assessing E. herbicola collection efficiency because very few culturable cells were obtained from surface samples. QPCR demonstrated that E. herbicola DNA was present in high concentrations on all of the surface samples, and sampling efficiencies ranged from 0.7 to 52.2%, depending on the sampling method and the surface material. The swab was generally more efficient than the sponge for collection of E. herbicola from surfaces. Test chamber trials were also performed in which E. herbicola was aerosolized into the chamber and allowed to settle onto test materials. Surface sampling results supported those obtained in laboratory trials. The results of this study demonstrate the capabilities of QPCR to enhance the detection and enumeration of biocontaminants on surface materials and provide information on the comparability of sampling methods.     

丝裂霉素C对Erwinia herbicola表达及运转冰核活性蛋白方式的影响

利用添加丝裂霉素C(MMC)于不同培养基中,并在不同培养条件下对草生欧文氏菌10025A(E．herbicola 10025A)诱导培养,首次证实该菌表达冰核活性蛋白的活性及运转分泌冰核活性蛋白方式是不同。研究结果显示,MMC的加入可以提高细菌培养物的冰核活性,该现象可以解释为MMC对E．herbicola的作用能够激活细胞SOS应急系统的反应,诱导细胞合成部分参与修复DNA损伤的酶和蛋白因子,达到对E．herbicola诱导表达冰核蛋白通过高尔基体形成小液泡,向膜外分泌的方式,同时对E．herbicola细胞分裂后的形态也发生明显的影响。这对研究细胞在恶劣环境的生存机制以及获取该条件下的蛋白具有重要的意义,对低温生物的研究也将产生积极地影响。     

Mechanisms of inhibition of Erwinia amylovora by Erw. herbicola in vitro and in vivo

The mechanisms by which Erwinia herbicola inhibits Erwinia amylovora , the fire blight pathogen, were investigated. The optimum pH for growth of Erw. amylovora strain Ea273 in nutrient-yeast extract-glucose broth (NYGB) was 7.0 and growth was markedly reduced at pH values below 6.0. In contrast, the growth rates of Erw. herbicola strains Eh141 and Eh112Y were only slightly reduced at pH levels as low as 4.5, relative to pH 6-8. When Ea273 was grown in NYGB in the presence of Eh141 or Eh112Y, the media became acidic and lower populations of Ea273 were recovered, compared with populations from buffered NYGB. Acidification of plant tissue as a consequence of growth of Erw. herbicola did not occur, however, and thus acid-based inhibition of growth in planta is unlikely. The growth rates of nine strains of Erw. herbicola and their abilities to reduce the pH of NYGB did not correlate with their different abilities to prevent development of fire blight incited by Ea273 in a research apple orchard. When grown in mixed culture, Eh114 and Eh112Y grew to higher populations than Ea273 due to depletion of a nitrogen source needed by Ea273. The ability of 12 strains of Erw. herbicola to produce antibiotics inhibitory to Ea273 on a glucose-asparagine medium correlated with the effectiveness of the strains in suppressing fire blight. A crude preparation of the Eh318 antibiotic delayed development of disease in immature pear fruits incited by Ea273 but not by strain Ea273R318, which is resistant in vitro to the Eh318 antibiotic. Cells of Eh318 protected immature pear fruits more effectively from infection by Ea273 than from the resistant strain Ea273R318.     

Characterization and plasmid profile of an inhibitory strain of Erwinia herbicola isolated from Phaseolous vulgaris in Egypt

Erwinia herbicola strain 48 was isolated from diseased phaseolous seedlings and characterized by biochemical properties, cellular fatty acid analysis and SDS-PAGE of the soluble cell protein. Although cellular fatty acid profile and the soluble cellular protein pattern showed high degree of similarity in comparison to those from E. herbicola strain 347417, obtained from the International Mycological Institute U.K., plasmid profiles were different. Both strains harbor a 23.1 kb plasmid, in addition, E. herbicola 48 contains 2 more plasmids (26.8 and 32.5 kb). The antagonism of E. herbicola 48 against a number of Gram-negative and Gram-positive bacteria was tested in vitro. Only Gram-negative bacteria were inhibited, suggesting that the inhibitory factor is likely to be bacteriocin.     

Intramolecular signal transmission in enterobacterial aspartate transcarbamylases II. Engineering co-operativity and allosteric regulation in the aspartate transcarbamylase of Erwinia herbicola

The aspartate transcarbamylase (ATCase) from Erwinia herbicola differs from the other investigated enterobacterial ATCases by its absence of homotropic co-operativity toward the substrate aspartate and its lack of response to ATP which is an allosteric effector (activator) of this family of enzymes. Nevertheless, the E. herbicola ATCase has the same quaternary structure, two trimers of catalytic chains with three dimers of regulatory chains ((c3)2(r2)3), as other enterobacterial ATCases and shows extensive primary structure conservation. In (c3)2(r2)3 ATCases, the association of the catalytic subunits c3 with the regulatory subunits r2 is responsible for the establishment of positive co-operativity between catalytic sites for the binding of aspartate and it dictates the pattern of allosteric response toward nucleotide effectors. Alignment of the primary sequence of the regulatory polypeptides from the E. herbicola and from the paradigmatic Escherichia coli ATCases reveals major blocks of divergence, corresponding to discrete structural elements in the E. coli enzyme. Chimeric ATCases were constructed by exchanging these blocks of divergent sequence between these two ATCases. It was found that the amino acid composition of the outermost beta-strand of a five-stranded beta-sheet in the effector-binding domain of the regulatory polypeptide is responsible for the lack of co-operativity and response to ATP of the E. herbicola ATCase. A novel structural element involved in allosteric signal recognition and transmission in this family of ATCases was thus identified.     

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).     

一株新的溶磷草生欧文氏菌的分离、鉴定及其溶磷效果的初步研究

从湖南、湖北、云南等地磷矿开采场的土壤样品中筛选到一株溶磷能力较强的菌株P21,结合生理生化指标和16S rDNA序列分析鉴定其属于草生欧文氏菌菠萝变种(Erwinia herbicola var.ananas).该菌能溶解磷酸三钙、羟基磷灰石、磷酸铁、磷酸锌,其中对磷酸三钙和羟基磷灰石的每升液体培养基溶磷量(P2O5)分别高达1206.20mg、529.67mg.溶磷菌草生欧文氏菌菠萝变种P21对产地不同的8种磷矿石溶解能力不同,对云南晋宁和昆阳、四川雅安、江苏锦屏等地磷矿石有较强的溶解能力,每升液体培养基溶磷量分别为96.64mg、78.46mg、67.07mg、65.24mg,对其它产地的磷矿石溶解能力较差.实验表明,培养液的pH下降与溶磷菌P21的溶磷量无直接关系.     

The presence of diverse IS elements and an avrPphD homologue that acts as a virulence factor on the pathogenicity plasmid of Erwinia herbicola pv. gypsophilae

The pathogenicity of Erwinia herbicola pv. gypsophilae (Ehg) and Erwinia herbicola pv. betae (Ehb) is dependent on a native plasmid (pPATH(Ehg) or pPATH(Ehb)) that harbors the hrp gene cluster, genes encoding type III effectors, phytohormones, biosynthetic genes, and several copies of IS1327. Sequence analysis of the hrp-flanking region in pPATH(Ehg) (cosmid pLA150) revealed a cluster of four additional IS elements designated as ISEhel, ISEhe2, ISEhe3, and ISEhe4. Two copies of another IS element (ISEhe5) were identified on the upstream region of the indole-3-acetic acid operon located on the same cosmid. Based on homology of amino acids and genetic organization, ISEhe1 belongs to the IS630 family, ISEhe2 to the IS5 family, ISEhe3 and ISEhe4 to different groups of the IS3 family, and ISEhe5 to the IS1 family. With the exception of ISEhe4, one to three copies of all the other IS elements were identified only in pathogenic strains of Erwinia herbicola pv. gypsophilae and Erwinia herbicola pv. betae whereas ISEhe4 was present in both pathogenic and nonpathogenic strains. An open reading frame that exhibited high identity (89% in amino acids) to AvrPphD of Pseudomonas syringae pv. phaseolicola was present within the cluster of IS elements. An insertional mutation in the AvrPphDEh, reduced gall size in gypsophila by approximately 85%. In addition, remnants of known genes from four different bacteria were detected on the same cosmid.     

Release of cell-free ice nuclei by Erwinia herbicola.

Several ice-nucleating bacterial strains, including Erwinia herbicola, Pseudomonas fluorescens, and Pseudomonas syringae isolates, were examined for their ability to shed ice nuclei into the growth medium. Only E. herbicola isolates shed cell-free ice nuclei active at -2 to -10 degrees C. These cell-free nuclei exhibited a freezing spectrum similar to that of ice nuclei found on whole cells, both above and below -5 degrees C. Partially purified cell-free nuclei were examined by density gradient centrifugation, chemical and enzymatic probes, and electron microscopy. Ice-nucleating activity in these cell-free preparations was associated with outer membrane vesicles shed by cells and was sensitive to protein-modifying reagents.     

The aroQ-encoded monofunctional chorismate mutase (CM-F) protein is a periplasmic enzyme in Erwinia herbicola.

Enteric bacteria possess two species of chorismate mutase which exist as catalytic domains on the amino termini of the bifunctional PheA and TyrA proteins. In addition, some of these organisms possess a third chorismate mutase, CM-F, which exists as a small monofunctional protein. The CM-F gene (denoted aroQ) from Erwinia herbicola was cloned and sequenced for the first time. A strategy for selection by functional complementation in a chorismate mutase-free Escherichia coli background was devised by using a recombinant plasmid derivative of pUC18 carrying a Zymomonas mobilis tyrC insert which encodes cyclohexadienyl dehydrogenase. The aroQ gene is 543 bp in length, predicting a 181-residue protein product having a calculated molecular mass of 20,299 Da. The E. herbicola aroQ promoter is recognized by E. coli, and a putative sigma-70 promoter region was identified. N-terminal amino acid sequencing of the purified CM-F protein indicated cleavage of a 20-residue signal peptide. This was consistent with the monomeric molecular mass determined for the enzyme of about 18,000 Da. The native enzyme is a homodimer. The implied translocation of CM-F was confirmed by osmotic shock experiments which demonstrated a periplasmic location. Immunogold electron microscopy indicated a polar localization within the periplasm. Polyclonal antibody raised against E. herbicola CM-F did not cross-react with the CM-F protein from the closely related Serratia rubidaea, as well as from a number of other gram-negative bacteria. Furthermore, when the E. herbicola aroQ gene was used as a probe in Southern blot hybridizations with EcroRI digests of chromosomal DNA from S. rubidaea and other enteric organisms, no hybridization was detected at low stringency. Thus, the aroQ gene appears to be unusually divergent among closely related organisms. The deduced CM-F amino acid sequence did not exhibit compelling evidence for homology with the monofunctional chorismate mutase protein of Bacillus subtilis.     

Physical and chemical control of released microorganisms at field sites

An important consideration in the environmental release of a genetically engineered microorganism is the capability for reduction or elimination of microorganism populations once their function is completed or if adverse environmental effects are observed. In this study the decontamination treatments of burning and biocide application, alone and in combination with tilling, were evaluated for their ability to reduce populations of bacteria released on the phylloplane. Field plots of bush beans (Phaseolus vulgaris), sprayed with the bacterium Erwinia herbicola, received the following treatments: control; control + till; burn; burn + till; Kocide (cupric hydroxide); Kocide + till; Agri-Strep (streptomycin sulfate); and Agri-Strep + till. Leaves and soil from the plots were sampled -1, 1, 5, 8, 12, 15, 19, and 27 days after application of the decontamination treatments. Burning produced a significant reduction in the number of E. herbicola, whereas tilling, alone or in combination with the biocide treatments, stimulated a significant increase in E. herbicola populations, which persisted for several weeks. The individual treatments of the biocides, Kocide and Agri-Strep, produced a rate of decline in E. herbicola populations that did not significantly differ from that of the control treatment.