The bacterium Rhodopseudomonas sphaeroides can perform a range of exotic metabolic processes valuable to biotechnology, such as photosynthesis, nitrogen fixation, and hydrogen gas production. Also significant, in evolutionary terms, is the apparently close relationship of this prokaryote to the mitochondria of eukaryotic organisms. Despite the importance of this organism, very little is known about its most fundamental aspect - its genetic organization. Hence a study was undertaken which exploited the properties of certain conjugative plasmids and transposons, so as to enable chromosome transfer and mapping in R. sphaeroides strain RS630.
IncP and IncW group antibiotic resistance R-plasmids carrying transposons, were transferred from Escherichia coli to R. sphaeroides by conjugation. R-plasmids were not stably inherited within R. sphaeroides as evidenced by the rapid loss of antibiotic resistance phenotypes during approximately 35 generations of growth in the absence of antibiotic selection. This aided the isolation of strains which had lost the R-plasmid but retained a copy of a transposon it carried. A conjugative R-plasmid, when introduced into such a Tn5 or Tn501-bearing strain, acquired a copy of the transposon at a frequency close to 10-2 per plasmid retransferred. Using this strategy, the ability of Tn5 and Tn501 to transpose in R. sphaeroides was demonstrated, and novel plasmid::transposon combinations were constructed.
In preliminary tests for chromosome mobilization ability (Cma), most wild-type IncP and IncW plasmids tested failed to exhibit Cma in R. sphaeroides; so too did plasmids carrying either Tn10 or Tn5. In dramatic contrast, plasmid RPl when carrying transposon Tn501, facilitated recovery of recombinants at frequencies within the range 10-3 to 10-7 per donor cell depending on the marker selected.
The use of RPl::Tn501 to promote chromosomal transfer provided marker coinheritance data from which genetic linkage maps were deduced. Initially two linkage groups were detected. One included a cluster of histidine biosynthesis markers located close to a putative origin of chromosomal transfer. The other linkage group encompassed a streptomycin resistance marker; clusters of markers affecting glycine and aromatic amino acid biosynthesis, respectively; and most notably, a single cluster of markers affecting photosynthesis, located close to a putative transfer origin. The unity of the chromosomal linkage map was evidenced by the existence of markers, located distal to the putative transfer origin(s), which showed linkage to both the groups initially detected. These results were compatible with a model which assumed the existence of a single circular chromosome, mobilized by RPl::Tn501 in either orientation predominantly from an origin located between, and proximal to, the histidine and photosynthesis clusters.
The apparent preference for an origin located close to the histidine cluster was displayed by Tn501-bearing derivatives of plasmids RPl, R751, pSa and R388/ despite differences in the site and orientation of the Tn501 insert. Cma was not exhibited in cases where Tn501 insertion caused loss of plasmid transferability. While Cma was displayed by strains harbouring Tn501 in trans relative to a coresident conjugative plasmid, there was evidence to suggest this activity required fusion of Tn501 and the plasmid to yield the cis configuration.
Transposons related to Tn501, such as Tn1, Tn801, Tn4, Tn1721 and Tn1723, showed little or no ability to enhance Cma in R. sphaeroides. A remarkable exception was the resolvase-deficient transposon Tn813. The derived element R751::Tn813 promoted the formation of recombinant classes distinct from, yet compatible with, those obtained using RPl::Tn501; and so provided data which complemented the original mapping studies.
Various plasmid::transposon combinations were tested for the ability to facilitate transfer of a coresident "non-mobilizable" cloning vector such as pBR322, between E. coli strains. Vector transfer frequencies as high as 10-1 per donor cell were obtained using either RPl::Tn501 or R751::Tn813. Strains of E. coli harbouring R751::Tn813 donated a coresident cloning vector (pBR322 or pUC8) to R. sphaeroides strains at a frequency close to 10-3 per donor, and at a frequency 100-fold higher when a restriction-deficient R. sphaeroides recipient was employed. Within R. sphaeroides, inheritance of the cloning vector markers was only detected in association with those of R751::Tn813. This implied that maintenance of the narrow host-range vector within R. sphaeroides was reliant upon its cointegration with the broad host-range plasmid.
In conclusion, the exceptional cointegration-facilitating ability of certain transposons, when combined with the conjugal transfer proficiency of an IncP plasmid, affords a convenient means to promote transfer of unrelated genetic material. The exploitation of these properties has finally provided operational genetic systems applicable to Rhodopseudomonas sphaeroides strain RS630.