The phytopathogenic enterobacterium Erwinia chrysanthemi excretes multiple isozymes of the plant tissue-disintegrating enzyme, pectate lyase (PL). Genes encoding PL were cloned from E. chrysanthemi CUCPB 1237 into Escherichia coli HB101 by inserting Sau3A-generated DNA fragments into the BamHI site of pBR322 and then screening recombinant transformants for the ability to sink into pectate semisolid agar. Restriction mapping of the cloned DNA in eight pectolytic transformants revealed overlapping portions of a 9.8-kilobase region of the E. chrysanthemi genome. Deletion derivatives of these plasmids were used to localize the pectolytic genotype to a 2.5-kilobase region of the cloned DNA. PL gene expression in E. coli was independent of vector promoters, repressed by glucose, and not induced by galacturonan. PL accumulated largely in the periplasmic space of E. coli. An activity stain used in conjunction with ultrathin-layer isoelectric focusing resolved the PL in E. chrysanthemi culture supernatants and shock fluids of E. coli clones into multiple forms. One isozyme with an apparent pI of 7.8 was produced at a far higher level in E. coli and was common to all of the pectolytic clones. Activity staining of renatured PL in sodium dodecyl sulfate-polyacrylamide gels revealed that this isozyme comigrated with the corresponding isozyme produced by E. chrysanthemi. The PL isozyme profiles produced by different clones and deletion derivative subclones suggest that the cloned region contains at least two PL isozyme structural genes. Pectolytic E. coli clones possessed a limited ability to macerate potato tuber tissues.
Stemphylium lotn, a pathogen of a cyanogenc plant, possesses a cyanide-isensitive alternate respiratory pathway. In the absence of cytochrome inhibitors, the alternate system had only a minor role in resption. When S. loi was grown in medium amended with antimycin to block the cytochrome chain, the alternate system accounted for the total oxygen consumption ocited with respiration. Hydrogen cyanide is released from cyanogenic glucosides in birdsfoot trefoil (Lotus corniculatus L.) upon infection by Stemphylium loti (16). This fungus grows in the presence of relatively high concentrations of cyanide in vitro (3) and is more tolerant of cyanide than are other fungi (16). reported that cyanide tolerance in S. loti is induced upon exposure to cyanide and is due to the production of the enzyme formamide hydro-lyase which converts cyanide to nontoxic formamide.We Cyanide-insensitive respiration is generally associated with an unidentified alternate terminal oxidase (ATO) (8) which reportedly connects with the normal electron transport chain at ubiquinone (24). This alternate respiratory pathway coexists with the normal respiratory chain in mitochondria (8). Studies of the normal and alternate respiratory systems have shown the following: (a) antimycin, cyanide, azide, and carbon monoxide, through inhibition of certain Cyt in the normal electron transport chain, inhibit the normal respiratory system but not the alternate pathway (9, 14, 25); (b) salicylhydroxamic acid specifically inhibits the alternate pathway but not the normal pathway (20); and (c) the addition of Cyt inhibitors to a growth medium promotes the appearance of the alternate respiratory pathway in microorganisms such that 100% of the respiration is insensitive to cyanide but is sensitive to SHAM (6,10,12).Our objectives were to determine if S. loti possesses a cyanideinsensitive alternate respiratory pathway and to assess the contribution of such an alternate pathway to growth and to increases in FHL activity. A preliminary report of this work has been published (17). MATERIALS AND METHODS
A chemical factor from wheat stem rust uredospores that induces infection structure formation has been identified as acrolein (2-propenal). This compound is the active component of distillates from uredospore extracts previously shown to induce infection structure formation.
The susceptibility of six spiroplasma strains to heavy-metal salt was characterized in terms of minimal inhibitory concentrations and minimal biocidal concentrations in broth tube dilution tests. The strains were most susceptible to mercuric chloride and silver nitrate; less susceptible to copper sulfate, cobalt chloride, lead nitrate, and cadmium sulfate; and least susceptible to nickel chloride and zinc sulfate. Spiroplasma citri strains Maroc R8A2 and C189 were the most susceptible to five of eight heavy-metal salts, and honeybee spiroplasma strain AS576 and Spiroplasma floricola strain 23-6 were generally the least susceptible. The difference between the minimal biocidal concentrations and the minimal inhibitory concentrations was greater for certain heavy-metal salts than for others.Spiroplasmas are motile, helical, cell wall-free procaryotes which have been classified as Mollicutes (14). Subsequent to their discovery in 1972 in association with stunted corn plants (12), they have been shown to induce some diseases of plants and insects, to induce disease experimentally in suckling rodents, and to be associated with apparently healthy plants, insects, and ticks (23,27, 29). The ability to cultivate these organisms in vitro permitted a dramatic increase in research on taxonomy, habitats and distribution, growth conditions, vector relations, antibiotic sensitivities, and morphological, biochemical, and physiological properties (26)(27)(28). Taxonomic studies have shown that spiroplasma strains can be separated into distinct groups and subgroups (6,10,16,17), and it has been suggested (9) that major groups as well as some distinct subgroups could be designated as separate species of the genus Spiroplasma. Three species, Spiroplasma citri, Spiroplasma floricola, and Spiroplasma mirum, of this genus have been described thus far (11,22,25).Nothing is known about the nature of inheritance in spiroplasmas, and little is known about the genome except for guanosine + cytosine contents (3,5,16,17,22), molecular weight (3,17,22), and the presence of plasmids in some strains (1, 21). Basic knowledge of spiroplasma genetics necessitates the development of a genetic system, which in turn requires a stock of mutant strains that provide genomic markers to follow and quantify movement and recombination of the genome.
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