Chloramphenicol acetyltransferase expression in marineRhodobacter sp. NKPB 0021 by use of shuttle vectors containing the minimal replicon of an endogenous plasmid

Matsunaga, Tadashi; Tsubaki, Kazufumi; Miyashita, Hideaki; Burgess, J. Grant

doi:10.1016/0147-619x(90)90011-z

Cited by 11 publications

(11 citation statements)

References 24 publications

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“…On the other hand, shuttle vectors for the marine photosynthetic bacterium Rhodobacter marinus have been constructed by connecting an endogenous plasmid to an Escherichia coli cloning vector, because broad-host-range vectors like those mentioned above are ineffective in this organism (5,32,33). These plasmids have not been demonstrated to function in other PNSB.…”

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confidence: 99%

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

Inui

Roh

Zahn

et al. 2000

Appl Environ Microbiol

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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...

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confidence: 99%

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

Inui

Roh

Zahn

et al. 2000

Appl Environ Microbiol

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“…The marine non sulfur photosynthetic bacterium Rhodobacter sp. strain NKB0021 was grown photosynthetically in modified RCV medium and has been described previously (Matsunaga et al, 1990). pUCl8/19 (Yanisch-Perron et al, 1985) were obtained commercially.…”

Section: Methodsmentioning

confidence: 99%

“…Plasmid pYGHPA12 (Watahiki et al, 1988) containing cloned ytGH cDNA was a gift from Professor K. Nakashima of Mie University, Japan. The shuttle vector pUK319 was constructed as described previously (Matsunaga et al, 1990) using pUC19 instead of pUC18. All plasmid constructions were verified by agarose gel electrophoresis (Sambrook et al, 1989).…”

Section: Methodsmentioning

confidence: 99%

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Expression of yellow tail (Seriola quinqueradiata) fish growth hormone cDNA in the marine photosynthetic bacterium Rhodobacter SP NKPB 0021

1993

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“…We previously screened small cryptic plasmids from marine Rhodobacter isolates and reported the construction of stable shuttle vectors containing the minimal replicon from a marine Rhodobacter isolate (10). In this study, a new cryptic plasmid designated pMGT was also found in M. magneticum MGT-1.…”

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Design and Application of a New Cryptic-Plasmid-Based Shuttle Vector for Magnetospirillum magneticum

Okamura

Takeyama

Sekine

et al. 2003

Appl Environ Microbiol

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A 3.7-kb cryptic plasmid designated pMGT was found in Magnetospirillum magneticum MGT-1. It was characterized and used for the development of an improved expression system in strain AMB-1 through the construction of a shuttle vector, pUMG. An electroporation method for magnetic bacteria that uses the cryptic plasmid was also developed.Magnetic bacteria synthesize intracellular, single-domain, nanometer-sized particles of magnetite (1) enveloped by lipid membranes that include numerous proteins. Aqueous dispersion of bacterial magnetic particles (BMPs) allows the development of highly sensitive chemiluminescence-based enzyme immunoassays by the chemical coupling of antibodies onto BMP surfaces (4). We have also previously reported applications of recombinant BMPs as novel bioassay platforms for protein display (12) and diagnosis of diabetes (19,20). The enormous potential of BMPs for nanobiotechnological applications calls for the development of cloning vectors for magnetic bacteria to allow convenient gene transfer and maximum expression.We have previously isolated two facultatively anaerobic Magnetospirillum magneticum strains, AMB-1 (ATCC 700264) (7) and MGT-1 (FERM P-16617) (8). The gene transfer system in M. magneticum AMB-1 magnetic bacteria was reported by our group (5). We have performed transposon mutagenesis in order to elucidate the molecular mechanism of BMP formation (11, 22). Electroporation has not been successfully applied in magnetic bacteria. Conjugative gene transfer was achieved (9) by using the broad-host-range vector pRK415 (3), but a low copy number was obtained. Replicons in the cryptic plasmids of magnetic bacteria may provide stable vectors yielding high copy numbers.In the photosynthetic bacterium Rhodospirillum rubrum, which is genetically close to Magnetospirillum sp., broad-hostrange vectors were unstable for replication (14). We previously screened small cryptic plasmids from marine Rhodobacter isolates and reported the construction of stable shuttle vectors containing the minimal replicon from a marine Rhodobacter isolate (10). In this study, a new cryptic plasmid designated pMGT was also found in M. magneticum MGT-1. This is the first report of a small-sized cryptic plasmid in magnetic bacteria. pMGT is extremely stable, even under nonselective conditions. This characteristic is valuable for the construction of shuttle vectors.The strains and plasmids used in this study are described in Table 1. The complete nucleotide sequence of pMGT (submitted to the DDBJ database) was 3,741 bp, with a GϩC content of 59.2%, which is lower than that of the Magnetospirillum genome (62 to 65%). Two potential open reading frames (ORF1 and ORF2) were found. ORF1 (positions 1727 to 2554 relative to position 1 at the BamHI site) is 828 bp in size and encodes a predicted protein of 275 amino acids (30.4 kDa). The ShineDalgarno (AAG, AGG, or GGA) sequences (16) were at positions 1711 to 1713. The deduced amino acid sequence showed significant homologies to the sequences of the Rep proteins in

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Chloramphenicol acetyltransferase expression in marineRhodobacter sp. NKPB 0021 by use of shuttle vectors containing the minimal replicon of an endogenous plasmid

Cited by 11 publications

References 24 publications

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

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

Expression of yellow tail (Seriola quinqueradiata) fish growth hormone cDNA in the marine photosynthetic bacterium Rhodobacter SP NKPB 0021

Design and Application of a New Cryptic-Plasmid-Based Shuttle Vector for Magnetospirillum magneticum

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