Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes

Abstract

Using physical and genetic data, we have demonstrated that Rhizobium meliloti SU47 has a symbiotic megaplasmid, pRmeSU47b, in addition to the previously described nod-nif megaplasmid pRmeSU47a. This plasm'id includes four loci involved in exopolysaccharide (exo) synthesis as well as two loci involved in thiamine 1'iosynthesis. Mutations at the exo loci have previously been shown to result in the formation of nodules which lack infection threads (Inf)' and fail to fix nitrogen (Fix-). Thus, both megaplasmids contain genes involved in the formation of nitrogen-fixing root nodules. Mutations at two other exo loci were not located on either megaplasmid. To mobilize the megaplasmids, the oriT of plasmid RK2 was inserted into them. On alfalfa, Agrobacterium tumefaciens strains containing pRmeSU47a induced marked root hair curling with no infection threads and Fix-nodules, as reported by others. This plant phenotype was not observed to change with A. tumefaciens strains containing both pRmeSU47a and pRmeSU47b megaplasmids, and strains containing pRmeSU47b alone failed to curl root hairs or form nodules.A great many natural isolates of rhizobia, agrobacteria, and pseudomonads have been shown to carry a variety of large plasmids (for examples, see references 8, 21, and 38). Some of these are megaplasmids, with molecular masses over 450 megadaltons (38). Genes for a few functions have been localized to these large plasmids, notably including pathogenicity gepes for the Ti and Ri plasmids of agrobacteria (6, 21) and symbiotic nodulation (nod) and nitrogen fixation (nif) genes for the Sym megaplasmids of the fast-growing rhizobia (3,8,28,33,37,38). Nevertheless, the significance of this genomic organization remains obscure, although the fact that these are all plant-associated soil bacteria does suggest an underlying evolutionary cause.Rhizobial Sym megaplasmids are being characterized extensively with regard to symbiosis. In addition, Rhizobium meliloti 41 has recently been shown to carry a second megaplasmid, with a molecular weight very nearly that of pSym, on which a region for surface exclusion (although none for symbiotic functions) has tentatively been identified (2). A second megaplasmid has also been identified in R.

A membrane metalloprotease participates in the sequential degradation of a Caulobacter polarity determinant

“…D-Glucose or L-arabinose (0.2%) was added to repress or induce, respectively, the expression of genes under the control of the P BAD promoter (Guzman et al, 1995). Plasmids were mobilized from E. coli to C. crescentus by bacterial conjugation with the help of pRK600 (Finan et al, 1986).…”

The role of the Serratia marcescens SdeAB multidrug efflux pump and TolC homologue in fluoroquinolone resistance studied via gene-knockout mutagenesis

“…The disrupted sdeB and sdeR fragments were transferred into the pKNG101 replacement vector and cloned into E. coli CC118 in a similar fashion as for hasF. Conjugation between the E. coli strain harbouring each of the disrupted S. marcescens sdeB, hasF and sdeR genes and the wild-type S. marcescens strain was carried out using E. coli helper strain MT616 (Finan et al, 1986) to create SDEAB1, HASF100 and SDER1, respectively. Conjugation was also carried out between the E. coli strain harbouring each of the disrupted S. marcescens sdeB and hasF genes and the S. marcescens clinical isolate strain (T-861), using E. coli helper strain MT616 (Finan et al, 1986) for comparison purposes.…”

“…Conjugation between the E. coli strain harbouring each of the disrupted S. marcescens sdeB, hasF and sdeR genes and the wild-type S. marcescens strain was carried out using E. coli helper strain MT616 (Finan et al, 1986) to create SDEAB1, HASF100 and SDER1, respectively. Conjugation was also carried out between the E. coli strain harbouring each of the disrupted S. marcescens sdeB and hasF genes and the S. marcescens clinical isolate strain (T-861), using E. coli helper strain MT616 (Finan et al, 1986) for comparison purposes. For the double sdeB/hasF-deficient mutant (SDEAB3/HASF300), conjugation was carried out between the S. marcescens sdeB (SDEAB1)-and hasF (HASF100)-deficient strains, using E. coli MT616 as a helper.…”

Pulsed-field gel electrophoresis studies of Rhizobiaceae

“…Interestingly, the size of the g. iaponicum genes for an essential biochemical pathway, the thiamine biosynthesis pathway, have been localized to a megaplasmid in R. meliloti (Finan et al, 1986). I believe that R. meliloti 1021 probably has three essential replicons in its genome, which should be called chromosomes, by definition, and that there is a large degree of genetic exchange between these replicons (Burkardt et al, 1987).…”

“…meliloti (Finan et al, 1986). Based on electron microscope (EM) contour measurements of large samples of megaplasmids, Burkardt et al (1987) Although E-meliloti is the most thoroughly studied of the…”