טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentKedar Tamar
SubjectDiversity in Extrachromosomal DNA Species in Several
Rhodococcus Erythropolis Strains
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Yechezkel Kashi


Abstract

             The phenotypic diversity exhibited by bacteria is a reflection of genotypic differences. Bacterial genomes may vary in the geometry, size and number of their chromosome(s), nucleotide distribution, gene order, and type and number of accessory elements. 

In this work, analyses for the genome of strains from the genus Rhodococcus were preformed. Some of the strains have catabolic phenotype of desulfurization (DBT+), which is encoded by a sequence termed as ‘sox operon’.

PFGE analysis for intact genomic DNA from 14 Rhodococcus strains in the Lehigh collection,  revealed the presence of extrachromosomal bands in eight of the strains. The nature of these bands is unknown. Therefore, these bands are termed ‘anomalous bands’.

In this work, the emphasis was on three R. erythropolis strains - 4277, 25544 and 53968 - that carry anomalous bands that migrate to the same position in pulsed-field gels. The objective of this work was to characterize the anomalous bands from strains 4277, 25544 and 53968, regarding their organization, size, geometry, association with sox operon, and the presence of rrn operon.

Through the restriction patterns of the anomalous bands it was demonstrated that the anomalous bands from strains 4277 and 25544 seem to be very similar, and different from the anomalous band from strain 53968.

            Estimates of size for the anomalous bands were accomplished based on two methods:  PFGE analysis and restriction fragments. It seems likely that the size of the anomalous bands from strains 4277 and 25544 is ~230 Kb, while the size of the anomalous band from strain 53968 is ~410 Kb.

Through a treatment with l exonuclease it was demonstrated that the behaviors of the anomalous bands from strains 4277, 25544 and 53968 are consistent with the behavior of a linear plasmid used as control.

 Based on the restriction results after AseI digest, XbaI digest, and double digest with AseI+XbaI,  restriction maps for the anomalous bands from strains 4277 and 25544 were obtained. The restriction maps indicate that the anomalous band from these two strains maybe linear molecule.

Through PCR amplification of regions within the sox operon, followed by Southern analysis, it was demonstrated that none of the anomalous bands carry the coding sequence for soxB gene, and among the anomalous bands from strains 4277, 25544 and 53968, only the latter was found to carry the IS elements found in the sox operon. 

Through PCR amplification of a region within 16S rDNA coding sequence, it was demonstrated that the anomalous bands from strains 4277, 25544 and 53968 do not carry rrn operons.