Mutants of Rhodopseudomonas sphaeroides useful in genetic analysis

Sistrom, W. R.; Macaluso, A; Pledger, Ralph J.

doi:10.1007/bf00413016

Cited by 30 publications

(12 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Complementation analyses have identified two cosmids which map to this region and which restore photosynthetic competence to MM1006 (unpublished results). Complementation with the R' plasmid, pWS2, derived from R. sphaeroides WS108 (36), also resulted in the restoration of photosynthetic competence to MM1006 (unpublished observations).…”

Section: Kaplan Unpublished Results)mentioning

confidence: 87%

Construction of TnphoA gene fusions in Rhodobacter sphaeroides: isolation and characterization of a respiratory mutant unable to utilize dimethyl sulfoxide as a terminal electron acceptor during anaerobic growth in the dark on glucose

Moore

Kaplan

1989

J Bacteriol

View full text Add to dashboard Cite

We have constructed a suicide vector, pUI800, containing the transposable element TnphoA (TnS ISSOL::phoA), for the purpose of producing protein fusions in vivo between the Escherichia coli alkaline phosphatase (APase) and proteins of the facultative photoheterotroph, Rhodobacter sphaeroides. We introduced TnphoA into the genome of R. sphaeroides at a coupled conjugation-transposition frequency of approximately 1 x 10-6. Fusions giving rise to APase expression, as judged by blue-colony pigmentation when exconjugants were plated on growth medium containing the chromogenic indicator 5-bromo-4-chloro-3-indolyl phosphate, were observed in about 1 % of the exconjugants. Numerous, distinguishable mutant phenotypes have been generated by this method, including those which lack the ability to use dimethyl sulfoxide as a terminal electron acceptor during anaerobic respiration, as well as those which are photosynthetically incompetent or altered in pigment synthesis, and others that express resistance to chlorate. The growth and spectral characteristics of several of these mutants, as well as the localization and quantitation of subcellular APase activity under different physiological conditions, have been examined. The presence of TnphoA in the host genome has been confirmed for each mutant analyzed, and specifically tagged DNA fragments containing TnphoA have been identified and localized; cosmids containing R. sphaeroides genomic DNA capable of complementing individual mutants have also been isolated. The usefulness of this approach in studying gene activity in R. sphaeroides is discussed.Rhodobacter sphaeroides is a purple, nonsulfur, facultative photoheterotrophic bacterium which is useful, as a model system, for the study of membrane biogenesis, bioenergetics, and photosynthesis. This organism is capable of chemoheterotrophic growth under aerobic conditions by using a variety of organic substrates; under anaerobic conditions in the light, cyclic electron flow provides the energetic capability for either photoautotrophic or photoheterotrophic growth (9,21 (9, 14); strains of E. coli were grown aerobically in either glucose-M9 minimal medium or LB broth (23). In the case of strain CC202, LB broth was supplemented with 0.2% lactose (wt/vol); low-phosphate SMM was prepared as previously described (9). When necessary, antibiotics were added to the following final concentrations: ampicillin, 25 ,ug/ml; tetracycline, 20 jig/ml in E. coli and 1 pLg/ml in R. sphaeroides; and kanamycin, 25 ,ug/ml.Conditions for anaerobic growth in the dark of R. sphaeroides on SMM containing glucose and DMSO, photoheterotrophic light intensities, and culture conditions have been reported by Donohue et al. (11). Anaerobic growth on SMM containing glucose and TMAO was accomplished by the substitution of 30 mM TMAO for DMSO in this medium (11). Resistance to chlorate (Chl') was assessed by the addition of 100 mM KCl03 to SMM.Conjugal matings. Conjugal matings between E. coli and R. sphaeroides were performed essentially as described by Davis et ...

show abstract

Section: Kaplan Unpublished Results)mentioning

confidence: 87%

Construction of TnphoA gene fusions in Rhodobacter sphaeroides: isolation and characterization of a respiratory mutant unable to utilize dimethyl sulfoxide as a terminal electron acceptor during anaerobic growth in the dark on glucose

Moore

Kaplan

1989

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…This plasmid was transformed into E. coli S17-1 and mobilized into R. sphaeroides 2.4.1 and CLia, and Smr/Spr Tcs double crossovers were isolated as previously described (17). Five (27,37) ( Fig. 9A, a) harboring approximately 109 kb of R. sphaeroides WS8 DNA containing the puf, puhA, cycA, and puc operons complemented CLla, restoring both the B800-850 complex and the normal carotenoid profile (Fig.…”

Section: Resultsmentioning

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

View full text Add to dashboard Cite

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...

show abstract

“…It has recently been suggested that carotenoids have an effect on the conformation of the B890 apoproteins from Rhodospirillum rubrum (4), suggesting that further studies are required to determine the basis for the pleiotropic nature of B800-850-deficient mutants in the formation of the photosynthetic membranes. The availability of the structural genes for the B800-850 complex (27) and the recent identification and isolation of the R. sphaeroides genes for carotenoid biosynthesis (35,39) will allow such mutants to be characterized at the molecular level.…”

Section: Discussionmentioning

confidence: 99%

Physiological and structural analysis of light-harvesting mutants of Rhodobacter sphaeroides

1988

View full text Add to dashboard Cite

Two mutants of Rhodobacter sphaeroides defective in formation of light-harvesting spectral complexes were examined in detail. Mutant RS103 lacked the B875 spectral complex despite the fact that substantial levels of the B875-oa polypeptide (and presumably the ,( polypeptide) were present. The B800-850 spectral complex was derepressed in RS103, even at high light intensities, and the growth rate was near normal at high light intensity but decreased relative to the wild type as the light intensity used for growth decreased. Mutant RS104 lacked colored carotenoids and the B800-850 spectral complex, as well as the cognate apoproteins. This strain grew normally at high light intensity and, as with RS103, the growth rate decreased as the light intensity used for growth decreased. At very low light intensities, however, RS104 would grow, whereas RS103 would not. Structural analysis of these mutants as well as others revealed that the morphology of the intracytoplasmic membrane invaginations is associated with the presence or absence of the B800-850 complex as well as of carotenoids. A low-molecular-weight intracytoplasmic membrane polypeptide, which may play a role in B800-850 complex formation, is described, as is a 62,000-dalton polypeptide whose abundance is directly related to light intensity as well as the absence of either of the light-harvesting spectral complexes. These data, obtained from studies of mutant strains and the wild type, are discussed in light of photosynthetic membrane formation and the abundance of spectral complexes per unit area of membrane. Finally, a method for the bulk preparation of the B875 complex from wild-type strain 2.4.1 is reported.

show abstract

Mutants of Rhodopseudomonas sphaeroides useful in genetic analysis

Cited by 30 publications

References 13 publications

Construction of TnphoA gene fusions in Rhodobacter sphaeroides: isolation and characterization of a respiratory mutant unable to utilize dimethyl sulfoxide as a terminal electron acceptor during anaerobic growth in the dark on glucose

Construction of TnphoA gene fusions in Rhodobacter sphaeroides: isolation and characterization of a respiratory mutant unable to utilize dimethyl sulfoxide as a terminal electron acceptor during anaerobic growth in the dark on glucose

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

Physiological and structural analysis of light-harvesting mutants of Rhodobacter sphaeroides

Contact Info

Product

Resources

About