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A derivative of Azospirillum brasilense Sp245, Sp245.5, which spontaneously lost 85 and 120 MDa replicons upon the formation of a new megaplasmid, has been shown to produce a novel lipopolysaccharide and to lose Calcofluor-binding polysaccharides. As compared to Sp245, the derivative displays notably increased heavy metal tolerance. The phenotypes of Sp245 and Sp245.5 are characterized by the following minimal inhibitory concentrations (MICs) of heavy metals: 0.5 and 0.9 μmol l(-1) of Ag(+), 0.4 and 0.7 mmol l(-1) of Co(2+), 0.9 and 4.7 mmol l(-1) of Cu(2+), and 3.1 and 11.5 mmol l(-1) of Zn(2+), respectively. In Sp245, in the presence of a nonlethal concentration (0.625 μmol l(-1)) of the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP), the MIC of cobalt, copper, and zinc drop 1.3- to 1.6-fold, but the low tolerance to silver is unaffected. In Sp245.5, CCCP does not affect cobalt tolerance, suppresses tolerance to copper and silver to the wild-type levels, and causes a 1.4-fold decrease in resistance to zinc. Therefore, significant elevation of heavy metal tolerance in Sp245.5 seems caused by the induction/overexpression of the proton-dependent efflux of certain metal ions. The novel cell surface and other unknown factors could also be responsible for the increased tolerance of A. brasilense Sp245.5 to heavy metals.

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Azospirillum brasilense swims in liquid environments and swarms in semisolid media. Five variants of A. brasilense Sp245, Sp245.P1-Sp245.P5, which swarmed faster than Sp245 in a semisolid malate-salt medium, have been isolated. In Sp245.P1-Sp245.P4, a new megaplasmid was revealed instead of an indigenous 85-MDa plasmid (p85). By polymerase chain reactions (PCR) with primers to the segments of p85 important for proper bacterial motility/ flagellation and for dissimilatory nitrite and NO reduction, that DNA of p85 was found retained by all the variants. In ERIC-and RAPD-PCR, microdiversity between the total DNAs of Sp245 and its variants was detected. Interstrain differences in growth characteristics in liquid peptonesuccinate-salt medium with KNO 3 or KNO 2 and in KNO 2 production/consumption were revealed. Although all the variants swam and swarmed faster than Sp245 in the medium supplemented with NH 4 Cl or KNO 3 , not all of them could do so in MPSS with KNO 2 .