Presence of a Family of Plasmids (29 to 65 Kilobases) with a 26-Kilobase Common Region in Different Strains of the Sulfur-Oxidizing BacteriumAcidithiobacillus caldus

Abstract

Three large cryptic plasmids from different isolates of Acidithiobacillus caldus were rescued by using an in vitro transposition system that delivers a kanamycin-selectable marker and an Escherichia coli plasmid origin of replication. The largest of the plasmids, the 65-kb plasmid pTcM1, was isolated from a South African A. caldus strain, MNG. This plasmid was sequenced and compared to that of pTcF1 (39 kb, from strain "f," South Africa) and pC-SH12 (29 kb, from strain C-SH12, Australia). With the exception of a 2.7-kb insertion sequence, pC-SH12 appears to represent the DNA common to all three plasmids and includes a number of accessory genes plus the plasmid "backbone" containing the replication region. The two larger plasmids carry, in addition, a number of insertion sequences of the ISL3 family and a composite transposon related to the Tn21 subfamily containing a highly mosaic region within the borders of the inverted repeats. Genes coding for arsenic resistance, plasmid mobilization, plasmid stability, and a putative restriction-modification system occur within these mosaic regions.

Architecture and Gene Repertoire of the Flexible Genome of the Extreme Acidophile Acidithiobacillus caldus

“…Typical plasmid replication, maintenance and/or mobilization gene modules were identified on the smaller plasmids ( Table 1 , Table S3b ). Plasmid pAca TY .1 resembles pLAtc2 and pLAtc3 and previously characterized members of the pTcM1 plasmid family [22] . These four plasmids share an 11 Kb region containing genes for replication, partitioning, a Pin-like invertase and an IncQ-like mobilization relaxase of the MobA-type, as well as small highly conserved proteins of unknown function.…”

“…Several aspects of its physiology have been studied in representative strains including sulfur oxidation [9] – [13] , central carbon metabolism [14] , resistance to arsenic, copper, iron and other heavy metals [15] – [20] and attachment and growth on minerals [21] . Also, a number of broad host range plasmids and a Tn21-like transposon have been characterized for the species [15] , [22] – [24] . According to these and other studies strain specific properties are apparent and further support the existence of divergent strain lineages within the A. caldus species.…”

Two Large, Related, Cryptic Plasmids from Geographically Distinct Isolates of Sulfobacillus thermotolerans

“…The finding that backbones of plasmids isolated from different strains of S. thermotolerans from different geographical locations are related but that the accessory genes are different appears to be a recurring theme in iron-and sulfuroxidizing bacteria. Species-specific plasmid backbones have been reported in Acidithiobacillus ferrooxidans (7,12,43), and Acidithiobacillus caldus (44). In A. caldus and S. thermotolerans, these plasmids appear to be either mobilizable or conjugable and may be involved in the movement of the accessory genes within isolates of the same species and possibly also between species.…”

“…In FeSYE medium, mineral salts (38; D. Johnson, personal communication) replace ordinary basal salts, and 0.025% yeast extract replaces tryptone soy broth. Plasmid DNA from S. thermotolerans strains L15 and Y0017 was prepared using a gentle lysis protocol followed by CsCl gradient centrifugation as previously described (44). Plasmids pL15 and pY0017 were "captured" and propagated in E. coli EC100D pir ϩ cells using the EZ::TN in vitro transposition system (EZ::TNϽR6K␥ori/KAN-2Ͼ transposon; Epicentre, Madison, WI) as previously described (44).…”

“…Plasmid DNA from S. thermotolerans strains L15 and Y0017 was prepared using a gentle lysis protocol followed by CsCl gradient centrifugation as previously described (44). Plasmids pL15 and pY0017 were "captured" and propagated in E. coli EC100D pir ϩ cells using the EZ::TN in vitro transposition system (EZ::TNϽR6K␥ori/KAN-2Ͼ transposon; Epicentre, Madison, WI) as previously described (44). A transposon containing a replicon capable of replication in E. coli pir ϩ cells and a kanamycin resistance gene was randomly inserted, and kanamycin-resistant E. coli transformants were screened using colony hybridizations with total digoxigenin-labeled pL15 as the probe (Roche).…”