Cloning and expression of bacterial ice nucleation genes in Escherichia coli.

Epiphytic populations of Pseudomonas syringae and Erwinia herbicola are important sources of ice nuclei that incite frost damage in agricultural crop plants. We have cloned and characterized DNA segments carrying the genes (ice) responsible for the ice-nucleating ability of these bacteria. The ice region spanned 3.5 to 4.0 kilobases and was continuous over this region in P. syringae Cit7R1. The cloned fragments imparted ice-nucleating activity in Escherichia coli. Substantial increases in the nucleating activity of both E. coli and P. syringae were obtained by subcloning the DNA fragments on multicopy plasmid vectors. Southern blot analysis showed substantial homology between the ice regions of P. syringae and E. herbicola, although individual restriction sites within the ice regions differed between the two species.     

Three separate classes of bacterial ice nucleation structures

Studies of the properties of the ice nucleation structure exposed on the surfaces of various bacteria such as Pseudomonas syringae, Erwinia herbicola, or various strains of Ice+ recombinant Escherichia coli have shown that there are clearly three major related but chemically distinct types of structures on these cells. First, the ability of Ice+ cells to nucleate super-cooled D2O has been examined, and it has been found that this ability (relative to the ability of the same cells to nucleate super-cooled H2O) exhibited three characteristic nucleating patterns. The rarest structure, called class A, is found on only a small fraction of cells in a culture, nucleates H2O at temperatures above -4.4 degrees C, and is an effective nucleator of super-cooled D2O. A second class of structure, called class B, is found on a larger portion of the cells, nucleates H2O between -4.8 and -5.7 degrees C, and is a relatively poor nucleator of super-cooled D2O. The class C structure is found on almost all cells and nucleates at -7.6 degrees C or colder. These three classes of structures were also differentiated by their sensitivities to low concentrations of water-miscible organic solvents such as dioxane or dimethyl sulfoxide. Depending on the specific bacterial strain, the addition of these solvents to bacterial suspensions lowered the nucleation activity of the class A structure by 1,000-fold or more. The nucleation activities of class B structures in the same culture were highly resistant to these compounds and were lowered only by 20 to 40%. The class C structures were more sensitive than Class B structures were, and the nucleation activities decreased 70 to 90%. Finally, the pH sensitivity of these three classes of structures was examined. The class A structure was destroyed in buffers at pH 4.5 lower but was stable in buffers at higher pHs. The class B structure was less sensitive to acidic buffers but was destroyed at pH 5.5 or lower and was stable at higher pHs. However, the class C structure was unaffected by incubation in buffers with pHs of 3.5 to 9.0. Suggestions for the actual nucleation structures of the three classes are proposed.     

冰核细菌表达冰核蛋白特性的研究

选用１００２５Ａ和ＱＦ－９５－Ｆ１９两株分离自杨树的冰核活性细菌,探讨了两株菌不同生长阶段与它们冰核活性表达的特性。实验结果显示,冰核活性细菌在ＭＰＤＡ培养液中表达冰核蛋白的特性及活性与细菌浓度、菌龄以及培养的环境条件相关,两株菌在表达冰核活性时对培养基的营养组分没有表现出特殊的要求。同时还进一步阐明了不同生长温度冰核活性细菌对冰核蛋白表达的影响。     

Immunological characterization of ice nucleation proteins from Pseudomonas syringae, Pseudomonas fluorescens, and Erwinia herbicola.

Antibodies were raised against the InaW protein, the product of the ice nucleation gene of Pseudomonas fluorescens MS1650, after protein isolation from an Escherichia coli clone. On Western blots (immunoblots), these antibodies recognized InaW protein and InaZ protein (the ice nucleation gene product of Pseudomonas syringae S203), produced by both E. coli clones and the source organisms. The InaZ protein appeared in P. syringae S203 during stationary phase; its appearance was correlated with the appearance of the ice nucleation-active phenotype. In contrast, the InaW protein occurred at relatively constant levels throughout the growth phases of P. fluorescens MS1650; the ice nucleation activity was also constant. Western analyses of membrane preparations of P. syringae PS31 and Erwinia herbicola MS3000 with this antibody revealed proteins which were synthesized with development of the nucleating phenotype. In these species the presence or absence of the nucleating phenotype was controlled by manipulation of culture conditions. In all nucleation-positive cultures examined, cross-reacting low-molecular-weight bands were observed; these bands appeared to be products of proteolytic degradation of ice nucleation proteins. The proteolysis pattern of InaZ protein seen on Western blots showed a periodic pattern of fragment sizes, suggesting a highly repetitive site for protease action. A periodic primary structure is predicted by the DNA sequence of the inaZ gene.     

Competitive Exclusion of Epiphytic Bacteria by IcePseudomonas syringae Mutants

The growth of ice nucleation-active and near-isogenic ice nucleation-deficient (Ice) Pseudomonas syringae strains coexisting on leaf surfaces was examined to determine whether competition was sufficient to account for antagonism of phylloplane bacteria. The ice nucleation frequency spectra of 46 IceP. syringae mutants, obtained after mutagenesis with ethyl methanesulfonate, differed both quantitatively and qualitatively, but the mutants could be grouped into four distinct phenotypic classes. The numbers of ice nucleation-active bacteria and ice nuclei active at -5 degrees C were reduced on plants colonized with IceP. syringae mutant strains before challenge inoculations with an IceP. syringae wild-type strain. Frost injury to plants pretreated with IceP. syringae strains was also reduced significantly compared with that to control plants and was correlated with the population size of the IceP. syringae strain and with the numbers of ice nuclei active at -5 degrees C. An IceP. syringae strain colonized leaves, flowers, and young fruit of pears in field experiments and significantly reduced the colonization of these tissues by IceP. syringae strains and Erwinia amylovora as compared with untreated trees.     

Formation of bacterial membrane ice-nucleating lipoglycoprotein complexes

The preliminary finding that nonprotein additions to the protein product of the ice-nucleating gene of Pseudomonas syringae or Erwinia herbicola are essential for ice nucleation at the warmest temperatures has led to experiments aimed at identifying possible linkages between the ice protein and the other components. It appears that the protein is coupled to various sugars through N- and O-glycan linkages. Mannose residues are apparently bound via an N-glycan bond to the amide nitrogen of one or more of the three essential asparagine residues in the unique amino-terminal portion of the protein. In turn, these mannose residues are involved in the subsequent attachment of phosphatidylinositol to the nucleation structure. This phosphatidylinositol-mannose-protein structure is the critical element in the class A nucleating structure. In addition to sugars attached to the asparagine residues, additional sugar residues appear to be attached by O-glycan linkages to serine and threonine residues in the primary repeating octapeptide, which makes up 70% of the total ice protein. These additional sugar residues include galactose and glucosamine and most likely additional mannose residues. These conclusions were based on (i) the changes in ice-nucleating activity due to the action of N- and O-glycanases, alpha- and beta-mannosidoses, and beta-galactosidase; (ii) immunoblot analyses of ice proteins in cell extracts after enzyme treatments; and (iii) the properties of transformed Ice+ Escherichia coli cells containing plasmids with defined amino-terminal and carboxyl-terminal deletions in the ice gene. Finally, evidence is presented that these sugar residues may play a role in aggregating the ice gene lipoglycoprotein compound into larger aggregates, which are the most effective ice nucleation structures.     

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。     

云南植物上冰核活性细菌鉴定

从云南植物上分离到９２株冰核活性细菌,并进行了鉴定。其中菠萝欧文氏菌６１株,占６６．３％;草生欧文氏菌２株,占２．２％;丁香假单胞菌２１株,占２２．８％;黄瓜角斑病菌２株,占２．２％;菜豆荚斑假单胞菌６株,占６．５％。云南省冰核活性细菌的优势种类是菠萝欧文氏菌,其次是丁香假单胞菌类。     

Localization of ice nucleation activity and the iceC gene product in Pseudomonas syringae and Escherichia coli

Ice nucleation activity and the iceC gene product were quantified in different subcellular fractions of the Pseudomonas syringae source strain and in Escherichia coli containing the cloned iceC gene to determine the activity of this protein in different subcellular locations. Ice nuclei were nearly completely retained during isolation of cell envelopes but exhibited a decrease in the temperature at which they were expressed. Ice nucleation activity was found in Triton X-100 insoluble membrane fragments as well as in slowly sedimenting and high-density membrane fragments. Nearly all ice nucleation activity was associated with the outer membrane because the partitioning of 3-ketodeoxyoctonate (a lipopolysaccharide component) and ice nuclei in cell fractions were similar to and opposite that of NADH oxidase (a cytoplasmic membrane component). The iceC gene product had an apparent mass of 150,000 Da based on migration in SDS-polyacrylamide gels. This protein was not found in soluble cell components. Nearly all of the iceC gene product, which occurred in low abundance, was associated with the outer membrane of both P. syringae and E. coli. Therefore, the iceC gene product is located at and is maximally active in or on the outer membrane of cells of the source strain and heterologous strains.     

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.     

UV-induced formation of filamentous forms in bacteria of Erwinia genus

Cells of 56 pectolytic Erwinia strains of different origin tested are prone to filamentation after UV-irradiation. The fact makes one possible to consider them natural fil+ organisms. Bacteria E. herbicola (9 strains) that are unable to synthesize pectatelyases are not transformed into filaments after NV-irradiation. The function of fil+ genes is recA-dependent in bacteria E. chrysanthemi ENA49 and is phenotypically analogous to fil+ gene function in E. coli B or lon- mutation in E. coli K12.     

Transmission of F'lac plasmid from Escherichia coli K-12 to bacteria of the genus Erwinia]

The F'lac plasmid was transferred by conjugation from Escherichia coli K-12 W1655 to 21 lac- strains of Erwinia spp. (5.2 . 10(-6) to 6.8 . 10(-2) lac+ exconjugants per donor cell). Erw. herbicola and Erw. chrysanthemi were the better recipients than others. The degree of the stability of lac+ genes in Erwinia exconjugants depends on the strains. Stable exconjugants of Erwinia, which harbored F'lac plasmid, were able to utilize lactose, to transfer lac genes by conjugation to Erwinia spp. and E. coli, and were sensitive to the F-specific phages f1, f2, Qbeta. The F'lac plasmid was eliminated from the exconjugants by the treatment with acridine orange, which indicates that this genetic element is not integrated into the Erwinia chromosome.     

Influence of Pathogenic and Non-pathogenic Bacteria on Soybean Suspension Cells

Co-cultivations of plant suspension cultures of soybean ( Glycine max ) with compatible phytopathogenic ( Pseudomonas syringae pv. glycinea ), incompatible phytopathogenic ( Pseudomonas syringae pv. tomato ), and different non-pathogenic ( Erwinia herbicola, Escherichia coli ) bacteria were carried out. Growth and viability (triphenyltetrazolium chloride activity) of plant cells and bacteria as well as enzyme activities of peroxidase (PO), polyphenoloxidase (PPO), and phenylalanine ammonialyase (PAL) within the plant cells were investigated over an incubation period of 7 days. The compatible pathogen inhibited growth and viability of the plant cells after 1 day and led to the death of the majority of the plant cells by the seventh day. In contrast, the incompatible pathogen directly reduced growth and viability of the soybean cells and caused a strong induction of enzyme activities of PO and PAL to more than 4 times of the untreated control by the seventh day. The epiphytic bacterium Erwinia herbicola caused a slight inhibition of growth and viability of the plant cells after the second day of co-cultivation. The PO activity increased in the same manner as in the incompatible interaction. The saprophytic Escherichia coli strain had a negligible influence on soybean suspension cells. All the bacteria tested except for Escherichia coli multiplied rapidly during co-cultivation and reached populations of 10⁸-10⁹ colonyfoming units/ml in the stationary phase. The results from this study demonstrate that the soybean suspension cells react differently to compatible, incompatible and saprophytic bacteria.     

Properties of a novel extracellular cell-free ice nuclei from ice-nucleating Pseudomonas antarctica IN-74

Some ice-nucleating bacterial strains, including Pantoea ananatis (Erwinia uredovora), Pseudomonas fluorescens, and Pseudomonas syringae isolates, were examined for the ability to shed ice nuclei into the growth medium. A novel ice-nucleating bacterium, Pseudomonas antarctica IN-74, was isolated from Ross Island, Antarctica. Cell-free ice nuclei from P. antarctica IN-74 were different from the conventional cell-free ice nuclei and showed a unique characterization. Cell-free ice nuclei were purified by centrifugation, filtration (0.45 microm), ultrafiltration, and gel filtration. In an ice-nucleating medium in 1 liter of cell culture, maximum growth was obtained with the production of 1.9 mg of cell-free ice nuclei. Ice nucleation activity in these cell-free ice nuclei preparations was extremely sensitive to pH. It was demonstrated that the components of cell-free ice nuclei were protein (33%), saccharide (12%), and lipid (55%), indicating that cell-free ice nuclei were lipoglycoproteins. Also, carbohydrate and lipid stains showed that cell-free ice nuclei contained both carbohydrate and lipid moieties.     

Ice nucleating activity of Pseudomonas syringae and Erwinia herbicola.

Chemical and biological properties of the ice nucleating sites of Pseudomonas syringae, strain C-9, and Erwinia herbicola have been characterized. The ice nucleating activity (INA) for both bacteria was unchanged in buffers ranging from pH 5.0 to 9.2, suggesting that there were no essential groups for which a change in charge in this range was critical. The INA of both bacteria was also unaffected by the addition of metal chelating compounds. Borate compounds and certain lectins markedly inhibited the INA of both types of bacterial cells. Butyl borate was not an inhibitor, but borate, phenyl borate, and m-nitrophenyl borate were, in order, increasingly potent inhibitors. These compounds have a similar order of affinity for cis hydroxyls, particularly for those found on sugars. Lentil lectin and fava bean lectin, which have binding sites for mannose or glucose, inhibited the INA of both bacteria. All other lectins examined had no effect. The inhibition of INA by these two types of reagents indicate that sugar-like groups are at or near the ice nucleating site. Sulfhydryl reagents were potent inhibitors of the INA of both bacteria. When treated with N-ethylmaleimide, p-hydroxymercuribenzoate, or iodoacetamide, the INA was irreversibly inhibited by 99%. The kinetics of inactivation with N-ethylmaleimide suggested that E. herbicola cells have at least two separate ice nucleating sites, whereas P. syringae cells have possibly four or more separate sites. The effect of infection with a virulent phage (Erh 1) on the INA of E. herbicola was examined. After multiple infection of a bacterial culture the INA was unchanged until 40 to 45 min, which was midway through the 95-min latent period. At that time, the INA activity began falling and 99% of the INA was lost by 55 min after infection, well before any cells had lysed. This decrease in INA before lysis is attributed to phage-induced changes in the cell wall.     

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.     

Acceptance and transfer of plasmid Rts1 by bacteria of the genus Erwinia]

The ability of 13 Erwinia strains to accept, to inherit and to transmit the Rts1 factor by conjugation was studied. 11 strains accepted the Rts1 factor from Escherichia coli K-12 CSH-2 with the frequency of about 10(-7)--10(-3). The Rts1 factor was genetically stable in the Erwinia cells and was not eliminated by acriflavine and under the temperature of 37 and 42 degrees C. All the R+ exconjugants were characterized with more high degree of the resistance of kanamycin than E. coli cells harbouring the same R factor. Erwinia strains harbouring the Rts1 plasmid transferred it by conjugation into homologic (Erwinia) and heterologic (E. coli) bacteria. The study of kinetics of the transfer of the Rts1 factor in different mating systems showed that the transfer of this plasmid from R+ Erwinia into R- Erwinia and R- E. coli--in the liquid medium. It is concluded that Erwinia can be the host and the donor of the Rts1 factor.     

Distribution of ice nucleation-active bacteria on plants in nature.

A replica plating method for rapid quantitation of ice nucleation-active (INA) bacteria was developed. Leaf washings of plant samples from California, Colorado, Florida, Louisiana, and Wisconsin were tested for the presence of INA bacteria. Of the 95 plant species sampled, 74 were found to harbor INA bacteria. Only the conifers were, as a group, unlikely to harbor INA bacteria. All of the INA bacteria isolated resembled either Pseudomonas syringae or Erwinia herbicola. Sufficient numbers of INA bacteria were present on the samples to account for the ice nuclei associated with leaves that are necessary for freezing injury to occur. Numbers of INA bacteria were large enough to suggest that plant surfaces may constitute a significant source of atmospheric ice nuclei.     

Expression of single chain antibodies (ScFvs) for c-myc oncoprotein in recombinant Escherichia coli membranes by using the ice-nucleation protein of Pseudomonas syringae

The ice nucleation protein (INP) is a glycosyl phosphatidylinositol anchored outer membrane protein found in certain Gram-negative bacteria. In this study, the INP from Pseudomonas syringae was applied as a fusion partner with the single chain antibody fragment (ScFv) against the human oncoprotein c-myc. Two new plasmids pNinaZ-myc and pNinaZScFv-BsaA1 were constructed and cloned into Escherichia coli JM109. The expression of the fusion protein was successfully demonstrated in the cloned cells. The fusion proteins had no effect on the viability of the host cells. Ice nucleation activity measurements and flow cytometry studies were followed to investigate the membrane expression of the fusion protein.