Localization of Low Copy Number Plasmid pRC4 in Replicating Rod and Non-Replicating Cocci Cells of Rhodococcus erythropolis PR4

Abstract

Rhodococcus are gram-positive bacteria, which can exist in two different shapes rod and cocci. A number of studies have been done in the past on replication and stability of small plasmids in this bacterium; however, there are no reports on spatial localization and segregation of these plasmids. In the present study, a low copy number plasmid pDS3 containing pRC4 replicon was visualized in growing cells of Rhodococcus erythropolis PR4 (NBRC100887) using P1 parS-ParB-GFP system. Cells were initially cocci and then became rod shaped in exponential phase. Cocci cells were found to be non-replicating as evident by the presence of single fluorescence focus corresponding to the plasmid and diffuse fluorescence of DnaB-GFP. Rod shaped cells contained plasmid either present as one fluorescent focus observed at the cell center or two foci localized at quarter positions. The results suggest that the plasmid is replicated at the cell center and then it goes to quarter position. In order to observe the localization of plasmid with respect to nucleoid, plasmid segregation was also studied in filaments where it was found to be replicated at the cell center in a nucleoid free region. To the best of our knowledge, this is the first report on segregation of small plasmids in R. erythropolis.

Rhodoccoccus erythropolis Is Different from Other Members of Actinobacteria : Monoploidy, Overlapping Replication Cycle, and Unique Segregation Pattern

“…The number of replisomes in LB-grown R. erythropolis cells shown previously using DnaB-GFP revealed one, two, three and four foci in cells (38). Since the terminus is the last region to be replicated, it is likely replicated at the cell center.…”

“…The Ptrc promoter has been previously shown to work in Rhodococcus (43). The plasmid pDS2 has been shown to be stable up to 60 generations (38). Transformants were selected on plates supplemented with streptomycin (100 g/ml), ampicillin (100 g/ml), and kanamycin (50 g/ml).…”

“…Fluorescence microscopy. The sample preparation for fluorescence microscopy was performed according to the protocol described by Singhi et al (38). Approximately 1 ml of exponentially growing cells was harvested at 2,500 rpm for 5 min and washed twice with 1 ml of 50 mM HEPES buffer (pH 7.4).…”

Functional characterization of the transcription regulator WhiB1 from Gordonia sp. IITR100

“…for 5 min, washed twice with 50 mM HEPES buffer (pH 7.5) and finally resuspended in 100 µl of the same buffer. About 2 µl of the washed cells was placed on a glass slide and observed under a microscope for observation of differences in cell morphology [18].…”

“…DAPI (5 µl, concentration 10 µg ml −1 ) was added to the cells and kept for 2 h in the dark. About 2 µl of the stained cells was placed on a glass slide and observed under the microscope to observe differences in chromosome segregation [18].…”