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

Abstract

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 ...

Regions of Rhodobacter sphaeroides cytochrome c2 required for export, heme attachment, and function

“…The precise cause of this degradation is unknown, but cleavage of other PhoA protein fusions has been observed in both R. sphaeroides (36,48) and E. coli (1,3,30,45). In fact, we see similar degradation of the CycA-PhoA hybrids in E. coli CC118 (data not shown), so it is apparently not related to the physiological function of cyt c2 in electron transport.…”

“…In addition, the preferential distribution of malate dehydrogenase activity in the cytoplasmic frac- tions confirms that significant amounts of cell lysis did not occur during shock fluid preparation. The low levels of alkaline phosphatase present in 2.4.1 and CYCAl (pC2P404.1) are due to the endogenous R. sphaeroides enzyme(s), which previous experiments have shown to be present in both periplasmic and cytoplasmic fractions (36,48).…”

“…Alternatively, the conformation of individual fusion proteins may reduce the ability of a given fusion to serve as a substrate for heme attachment or reduce the activity of that fusion in a heme peroxidase assay once heme is attached. However, it appears that this disparity is not due to titration of a cellular heme pool by plasmidencoded proteins, because copy number levels of cyt c2 are produced in cells that contain the intact cycA gene in trans (5 (36). Several of the fusion proteins are substrates for heme attachment, and one of them (Q119) is apparently able to function in electron transport.…”

“…The plasmids have been named pC2A11 to pC2Q119 according to the position of the cycA-phoA fusion, and we have designated gene products encoded by these plasmids as All, A30, etc. While wild-type R. sphaeroides is phenotypically pho (36,48), cells containing any of the five CycA-PhoA fusions exhibit a pho+ (blue) phenotype on 5-bromo-4-chloro-3-indolyl phosphate plates, suggesting that all of the hybrid proteins were exported. In addition, since a transcriptiontranslation stop cartridge was placed upstream of the cycA sequences, this result indicates that transcription of the fusions is directed by the R. sphaeroides cycA promoter sequences contained on the plasmid.…”

“…To analyze the location of the fusion proteins, R. sphaeroides shock fluid was prepared (46), and cytoplasmic, inner membrane, and outer membrane fractions were separated as described previously (46,49). Malate dehydrogenase assays (31) and reduced (ascorbate)-minus-oxidized (potassium ferricyanide) difference spectra (13,24) (36).…”