A self-transmissible, narrow-host-range endogenous plasmid of Rhodobacter sphaeroides 2.4.1: physical structure, incompatibility determinants, origin of replication, and transfer functions

Suwanto, Antonius; Kaplan, Samuel

doi:10.1128/jb.174.4.1124-1134.1992

Cited by 32 publications

(27 citation statements)

References 42 publications

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“…Conjugation to introduce plasmids from E. coli to R. sphaeroides was done as described previously (37). R. sphaeroides-R. sphaeroides matings were routinely performed under photosynthetic condition as follows.…”

Section: Methodsmentioning

confidence: 99%

“…DNA fragments isolated from agarose gels were routinely purified by using a Gene Clean Kit (Bio 101 Inc., La Jolla, Calif.). Southern hybridization analysis was performed as described previously (37), by using stringent washing conditions at 60°C twice for 15 min each time and detection by a chemiluminescence method (Photogene detection system; BRL) or direct exposure to X-ray film (Kodak X-OMAT) when using [o-32P]dCTPlabeled probes.…”

Section: Methodsmentioning

confidence: 99%

“…Escherichia coli and R. sphaeroides strains were routinely grown in LB (37) at 37°C and Sistrom's minimal medium lacking glutamate and aspartate, pH 7.2 (Sis), at 32°C (23), respectively, unless otherwise stated. An antibiotic(s) and sucrose were supplemented whenever appropriate in the following concentrations: kanamycin (Km), 25 ,ug/ml; spectinomycin (Sp), 50 ,ug/ml; streptomycin (Sm), 50 ,ug/ml; nalidixic acid (Nx), 20 ,ug/ml; rifampin, 50 ,ug/ml; tetracycline (Tc), 1 ,ug/ml (for R. sphaeroides) and 15 ,ug/ml (for E. coli); ampicillin, 150 ,ug/ml; chloramphenicol, 10 ,ug/ml.…”

Section: Methodsmentioning

confidence: 99%

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Chromosome transfer in Rhodobacter sphaeroides: Hfr formation and genetic evidence for two unique circular chromosomes

Suwanto

Kaplan

1992

J Bacteriol

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A 600-bp oriT-containing DNA fragment from the Rhodobacter sphaeroides 2.4.1 S factor (oriTs) (A. Suwanto and S. Kaplan, J. Bacteriol. 174:1124-1134, 1992) was shown to promote polarized chromosomal transfer when provided in cis. A Kmr-oriTs-sacR-sacB (KTS) DNA cassette was constructed by inserting oriTs-sacR-sacB into a pUTmini-Tn5 Km1 derivative. With this delivery system, KTS appeared to be randomly inserted into the genome of R. sphaeroides, generating mutant strains which also gained the ability to act as Hfr donors. An AseI site in the Kmr cartridge (from Tn903) and DraI and SnaBI sites in sacR-sacB (the levansucrase gene from Bacillus subtilis) were employed to localize the KTS insertion definitively by pulsed-field gel electrophoresis. The orientation of oriTs at the site of insertion was determined by Southern hybridization analysis. Interrupted mating experiments performed with some of the Hfr strains exhibited a gradient of marker transfer and further provided genetic evidence for the circularity and presence of two chromosomal linkage groups in this bacterium. The genetic and environmental conditions for optimized mating between R. sphaeroides strains were also defined. The results presented here and our physical map of the R. sphaeroides 2.4.1 genome are discussed in light of the presence of two chromosomes.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Chromosome transfer in Rhodobacter sphaeroides: Hfr formation and genetic evidence for two unique circular chromosomes

Suwanto

Kaplan

1992

J Bacteriol

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“…Southern hybridization analysis of genomic DNA was performed as described previously (3,5). Endogenous plasmid profiles of indicated bacterial strains following SpeI digestion were analyzed by TAFE (transverse alternating-field electrophoresis) gel analysis and compared with those of the wild-type strain (32). RNA isolation and Northern (RNA) hybridization.…”

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confidence: 99%

Isolation and characterization of trans-acting mutations involved in oxygen regulation of puc operon transcription in Rhodobacter sphaeroides

Lee

Kaplan

1992

J Bacteriol

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Transcriptional expression of the puc operon in Rhodobacter sphaeroides 2.4.1 is dependent on the partial pressure of oxygen. By using transcriptional fusions in trans of a promoterless fragment derived from the aminoglycoside-3'-phosphotransferase gene of Tn903 to puc operon-specific DNA containing a 629-bp 5' cis-acting regulatory region involved in the expression of puc-specific mRNA, we selected Kmr colonies under aerobic conditions. Two broad classes of mutations, trans and cis, which are involved in 02 control of puc operon transcription, fall into several distinct phenotypic classes. The cis-acting regulatory mutations are characterized in detail elsewhere (J. K. Lee and S. Kaplan, J. Bacteriol. 174:1146-1157, 1992. Two trans-acting regulatory mutants, CL1a and Tla, which are B800-850-Car-and apparently B875-, respectively, were shown to derepress puc operon transcription in the presence of oxygen. The mutation giving rise to CLia has been shown to act at the puc operon-specific cis-acting upstream regulatory region (-629 to -92).On the other hand, the mutation giving rise to Tla, identifying a second trans-acting regulatory factor(s), appears to act at both the upstream (-629 to -92) and the downstream (-92 to -1) regulatory regions of the puc operon as well as at the level(s) of bacteriochlorophyll and carotenoid biosyntheses, as revealed by the presence of the B800-850 complex under chemoheterotrophic growth conditions. Both the B800-850-Carphenotype and the trans-acting effect on puc operon expression in mutant CL1a were complemented with a 2.2-kb DNA fragment located within the carotenoid gene cluster. Mutant T1, was complemented with a 7.0-kb EcoRI restriction fragment containing the puhA gene and its flanking DNA (6.3 kb) to restore expression of the B875 complex and to suppress the trans-acting effect resulting in the loss of 02 control. Under chemoheterotrophic conditions, mutant Tla was highly unstable, segregating into a PS-mutant designated T4.The puc operon of Rhodobacter sphaeroides consists of the pucBA structural genes (encoding the B800-850-, and -ot polypeptides, respectively) and additional DNA sequences which extend approximately 1.8 kb immediately downstream and which encode a gene product(s) apparently involved in the posttranslational regulation or assembly of the pucBA gene products, resulting in the formation of the B800-850 light-harvesting complex (10,16,17). In a related bacterium, Rhodobacter capsulatus, involvement of the gene products encoded by the genes pucCDE immediately downstream of pucBA in the formation of the B800-850 complex has also been reported (35). When transcribed, the puc operon of R. sphaeroides yields 0.5-and 2.3-kb pucspecific transcripts. The transcripts share the same 5' end, which is localized 117 nucleotides upstream of the start of the pucB gene (17) regulation of puf operon expression (13). Klug and Jock (14) further suggested that the cis-acting mutation within the Q gene upstream region could result in an altered regulation of puc operon express...

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“…1), one Calvin cycle gene (cbbX), and ϳ50 uncharacterized genes encoding proteins of unknown function (Table 2). Of the uncharacterized genes in cluster 2, RSP4157 to RSP4164 were located on the self-transmissible 101-kb plasmid P004 (33). These genes were flanked by open reading frames (ORFs) predicted to encode transposase proteins (RSP4156 and RSP4165 to RSP4166), which were transcribed from the opposite strand and exhibited low or unchanged levels of RNA under all conditions analyzed (Fig.…”

Section: Resultsmentioning

confidence: 99%

Identification of Genes Required for Recycling Reducing Power during Photosynthetic Growth

2005

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Photosynthetic organisms have the unique ability to transform light energy into reducing power. We study the requirements for photosynthesis in the ␣-proteobacterium Rhodobacter sphaeroides. Global gene expression analysis found that ϳ50 uncharacterized genes were regulated by changes in light intensity and O 2 tension, similar to the expression of genes known to be required for photosynthetic growth of this bacterium. These uncharacterized genes included RSP4157 to -4159, which appeared to be cotranscribed and map to plasmid P004. A mutant containing a polar insertion in RSP4157, CT01, was able to grow via photosynthesis under autotrophic conditions using H 2 as an electron donor and CO 2 as a carbon source. However, CT01 was unable to grow photoheterotrophically in a succinate-based medium unless compounds that could be used to recycle reducing power (the external electron acceptor dimethyl sulfoxide (DMSO) or CO 2 ) were provided. This suggests that the insertion in RSP4157 caused a defect in recycling reducing power during photosynthetic growth when a fixed carbon source was present. CT01 had decreased levels of RNA for genes encoding putative glycolate degradation functions. We found that exogenous glycolate also rescued photoheterotrophic growth of CT01, leading us to propose that CO 2 produced from glycolate metabolism can be used by the Calvin cycle to recycle reducing power generated in the photosynthetic apparatus. The ability of glycolate, CO 2 , or DMSO to support photoheterotrophic growth of CT01 suggests that one or more products of RSP4157 to -4159 serve a previously unknown role in recycling reducing power under photosynthetic conditions. Life on earth is dependent upon photosynthesis, either directly using energy from sunlight or indirectly through the use of organic compounds produced by photosynthetic organisms. Due to the importance of photosynthesis, much research has been devoted to understanding this process in plants, algae, and photosynthetic bacteria. The defining feature of all photosynthetic organisms is their ability to use light energy to generate reducing power, which is used to support the synthesis of ATP, the assimilation of CO 2 , or the synthesis of other compounds. We are studying the requirements for photosynthesis in the ␣-proteobacterium Rhodobacter sphaeroides, a facultative phototroph that also can produce energy by aerobic or anaerobic respiratory pathways.Since the continual flow of electrons through electron carriers is critical to energy production, cells have evolved strategies to maintain necessary electron flow by recycling or disposing of excess reducing power. For example, fermentative pathways use organic compounds as electron acceptors, and electron transport to O 2 allows the aerobic respiratory chain to produce energy. When O 2 is absent, anaerobic respiratory pathways reduce alternate electron acceptors like dimethyl sulfoxide (DMSO) to dispose of excess reducing power (18). Photosynthetic growth also requires a strategy for recycling reducing power, but di...

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A self-transmissible, narrow-host-range endogenous plasmid of Rhodobacter sphaeroides 2.4.1: physical structure, incompatibility determinants, origin of replication, and transfer functions

Cited by 32 publications

References 42 publications

Chromosome transfer in Rhodobacter sphaeroides: Hfr formation and genetic evidence for two unique circular chromosomes

Chromosome transfer in Rhodobacter sphaeroides: Hfr formation and genetic evidence for two unique circular chromosomes

Isolation and characterization of trans-acting mutations involved in oxygen regulation of puc operon transcription in Rhodobacter sphaeroides

Identification of Genes Required for Recycling Reducing Power during Photosynthetic Growth

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