Mapping of RP4 plasmid using deletion mutants of pAS8 hybrid (RP4-ColE1)

Abstract

We used the hybrid plasmid pAS8 in order to conduct the genetic analysis of RP4 plasmid. The presence of two replicons in the hybrid plasmid permitted to expand the spectrum of deletion mutants of RP4 isolated, which are capable to autonomous replication. The shortening of the hybrid plasmid was achieved by P22 transduction, by induction of deletion mutants using mitomycin C, as well as by seletion of Tra- mutants on the basis of resistance of cells to P-specific phages. These techniques have lead to isolation of clones possessing different combinations of plasmid resistance determinants. Comparison of phenotypic characteristics of deletion plasmids pAS9, pAS10, pAS11, pAS12 and pAS10-2 permitted to propose the map for pAS8 plasmid with the following sequence of markers: tra--kam--ColE1--amp--tet... Heteroduplex analysis of deletion mutants of pAS8 permitted to construct a physical map and to elaborate in greater detail the functional map of RP4 plasmid. The correlation between the ability of mutants to replicate in polA(TS) strain at nonpermissive conditions and the length of the deleted segment permitted to map rep genes of RP4 on a region with coordinates 9.8--17.3 kb. A relationship between the manifestation of incompatibility of mutants with Inc P-1 plasmids and the length of deletions points out that inc genes are located on DNA region with coordinates 2.1--9.8 kb. The analysis of replication of deletion mutants and the manifestation of incompatibility just as of the data about the size of appropriate deletions permitted to make the conclusion about the functional and genetic independence of the replication control and incompatibility control in RP4 plasmid.

Analysis of copy number control elements in the region of the vegetative replication origin of the broad host range plasmid RK2.

“…It is possible that as yet uncharacterized control elements lie elsewhere on RK2. Indeed, other weakly acting incompatibility loci have been located (Sakanyan et al, 1978;Meyer and Hinds, 1982 region as deduced from the DNA sequence Waters, 1982;Waters et al, 1983 The way in which copA and copB act is as yet unknown. Both of these elements express incompatibility suggesting that they are not simply cis-acting functions.…”

“…It is possible that as yet uncharacterized control elements lie elsewhere on RK2. Indeed, other weakly acting incompatibility loci have been located (Sakanyan et al, 1978;Meyer and Hinds, 1982; K. Ellis and P. Barth, personal communication; A. Hussain and C.M. Thomas, in preparation), although these may reflect partitioning functions rather than replication control.…”

Two localization motifs mediate polar residence of FrzS during cell movement and reversals of Myxococcus xanthus

“…The pCT2 integration vector was constructed as follows: the tet repressor and promoter region from E. coli plasmid RP4 (Sakanyan et al, 1978) was flanked on each side by 1 kb M. xanthus DNA regions from the car locus encoding carotenoid biosynthetic enzymes (Lopez-Rubio et al, 2002) and cloned in pBJ113. frzS was then cloned in pCT2 immediately downstream from the tet promoter region.…”

Suicide vector for transposon mutagenesis in Pseudomonas solanacearum

“…Staskawicz (10) used the bacteriophage Mu-based suicide vector pJB4JI (2). We tested pJB4JI and other IncP-based suicide plasmids, such as pSUB2021 (9) and pAS8Rep-1 (8), and found that they transferred the transposon into most P. solanacearum strains at very low frequencies. The plasmid pJB4JI also showed a high frequency of survival, a problem that has been previously reported (4, 7).…”