Sequence Analysis of the Cryptic Plasmid pMG101 from Rhodopseudomonas palustris and Construction of Stable Cloning Vectors

Abstract

A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of ␣-proteobacteria.Purple nonsulfur bacteria (PNSB) are an assemblage of phenotypically diverse species. Under anaerobic conditions in the light, all species grow photoheterotrophically when supplied with various organic substrates or photoautotrophically with CO 2 as a sole carbon source. Under microaerobic to aerobic conditions in the dark, many representatives can grow chemoheterotrophically, and some grow chemoautotrophically (40).To develop a new CO 2 -fixing bioprocess, we have been performing biochemical and genetic analyses of intermediary metabolism, including CO 2 fixation, underlying the complex modes of growth in the PNSB, using Rhodopseudomonas palustris as a model microorganism (4,23,24). For this purpose, development of a versatile host-vector system would be helpful.In R. palustris and other PNSB, broad-host-range vectors have been used to provide the tools for gene transfer. The most widely used vectors are derivatives of RK2 such as pRK415 (25) and pLAFR1 (14). Cloning vector pRK415 has been utilized for genetic analyses of several R. palustris genes (8, 17), and cosmid vector pLAFR1 has been used to make a library of R. palustris DNA (8). However, these plasmids were unstable in R. palustris under nonselective conditions (M. Inui, unpublished data), as also observed in R. sphaeroides (9) and in Rhodospirillum rubrum (42).Other vectors derived from the broad-host-range plasmid RSF1010 such as pDSK519 (25) can also replicate in PNSB, including R. palustris (Inui, unpublished data). But this vector was also unstable under nonselective conditions in R. palustris and R. sphaeroides (Inui, unpublish...

Complete Nucleotide Sequence of a 43-Kilobase Genomic Island Associated with the Multidrug Resistance Region ofSalmonella entericaSerovar Typhimurium DT104 and Its Identification in Phage Type DT120 and Serovar Agona

“…The C-terminal region of the S025 product, which exhibits similarity to the N-terminal region of the Y4BN hypothetical protein of pNGR234a, contains a peptidase (S8) conserved domain from the subtilase family of proteinases. The S003 product exhibits similarity to the DNA replication protein RepA from the Rhodopseudomonas plasmid pMG101 (19). The S024 product is similar to the phage P2 OLD protein, which may be an exonuclease involved in overcoming defects in lysogenization (19).…”

“…The S003 product exhibits similarity to the DNA replication protein RepA from the Rhodopseudomonas plasmid pMG101 (19). The S024 product is similar to the phage P2 OLD protein, which may be an exonuclease involved in overcoming defects in lysogenization (19). One other ORF product, that of S020, showed similarity to a plasmid protein of unknown function, Orf3, from the Francisella plasmid pFNL10 (AF121418).…”

A periplasmic, α-type carbonic anhydrase from Rhodopseudomonas palustris is essential for bicarbonate uptake

“…7 strain does not show β-galactosidase activity, we constructed a plasmid which contains the E. coli lacZ gene using an Rps. palustris shuttle vector (Inui et al, 2000). To examine the localization of CA by another method we used immunoblot analysis.…”

Evolutionary relationship of phototrophic bacteria in the α-Proteobacteria based on farnesyl diphosphate synthase The GenBank accession numbers for the FPP synthase gene sequences reported in this paper are AB053173–AB053178, AB053180 and AB062882–AB062884.

“…They do not, however, constitute a phylogenetically homogeneous taxon based on 16S rRNA sequences, and are found in three of the five Proteobacteria classes, namely α-, β-and γ-Proteobacteria (Stackebrandt et al, 1996), intermingling with non-phototrophic bacteria. For example, Rhodopseudomonas palustris, a phototrophic purple non-sulfur bacterium belonging to the α-2 Proteobacteria, is phylogenetically close to the nodule-forming, non-phototrophic Bradyrhizobium japonicum (Inui et al, 2001 ;Fleischman et al, 1995 ;Wong et al, 1994 ;Young et al, 1991). Moreover, Rhodobacter species in …”