Characterization of a cryptic plasmid from a Greenland ice core Arthrobacter isolate and construction of a shuttle vector that replicates in psychrophilic high G+C Gram-positive recipients.
Over 60 Greenland glacial isolates were screened for plasmids and antibiotic resistance/sensitivity as the first step in establishing a genetic system. Sequence analysis of a small, cryptic, 1,950 bp plasmid, p54, from isolate GIC54, related to Arthrobacter agilis, showed a region similar to that found in theta replicating Rhodococcus plasmids. A 6,002 bp shuttle vector, pSVJ21, was constructed by ligating p54 and pUC18 and inserting a chloramphenicol acetyl transferase (CAT) cassette conferring chloramphenicol resistance. Candidate Gram-positive recipients were chosen among glacial isolates based on phylogenetic relatedness, relatively short doubling times at low temperatures, sensitivity to antibiotics, and absence of indigenous plasmids. We developed an electroporation protocol and transformed seven isolates related to members of the Arthrobacter, Microbacterium, Curtobacterium, and Rhodoglobus genera with pSVJ21. Plasmid stability was demonstrated by successive transformation into Escherichia coli and four Gram-positive isolates, growth without antibiotic, and plasmid re-isolation. This shuttle vector and our transformation protocol provide the basis for genetic experiments with different high G+C Gram-positive hosts to study cold adaptation and expression of cold-active enzymes at low temperatures.
Miteva
Vanya
V
Department of Biochemistry and Molecular Biology, 211 S. Frear, The Pennsylvania State University, University Park, PA 16802, USA. vim1@psu.edu
Lantz
Sarah
S
Brenchley
Jean
J
D000222:Adaptation, Physiological; D001173:Arthrobacter / Q000235:genetics* / Q000302:isolation & purification; D001482:Base Composition; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002702:Chloramphenicol Resistance / Q000235:genetics; D003080:Cold Temperature; D004261:DNA Replication; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005822:Genetic Vectors; D006094:Gram-Positive Bacteria / Q000235:genetics* / Q000254:growth & development; D006116:Greenland; D046448:Ice Cover / Q000382:microbiology*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids; D017422:Sequence Analysis, DNA; D013997:Time Factors; D014169:Transformation, Bacterial
D015500:Chloramphenicol O-Acetyltransferase