Transfer of kanamycin resistance mediated by plasmid R68.45 in Paracoccus denitrificans.

Plasmid R68.45 mediates the transfer of kanamycin resistance from Pseudomonas aeruginosa to Paracoccus denitrificans. Kanamycin resistance could be transferred from one strain of P. denitrificans to another, thus opening up the possibility of using R68.45 as a sex factor in P. denitrificans.     

Genetic transformation of Rhodopseudomonas sphaeroides by plasmid DNA.

A broad-host-range cloning vector, pUI81, was constructed in vitro from plasmids RSF1010 and pSL25 (a pBR322 derivative) and used to assay for transformation in Rhodopseudomonas sphaeroides. Washing cells with 500 mM Tris was an effective means of inducing competence for DNA uptake. Transformation frequencies as high as 10(-5) (transformants per viable cell) have been achieved by incubating Tris-treated cells with plasmid DNA, 100 mM CaCl2, and 20% polyethylene glycol 6000. Maximum frequencies were obtained when recipient cells were spread onto selective media after a 6.5-h outgrowth period in antibiotic-free medium. The structure (open circular versus closed, covalent circular), size, and concentration of plasmid DNA all significantly affected the transformation frequency. Four different plasmids, all small and suitable as cloning vectors, have been introduced by transformation into several different R. sphaeroides strains. Recombinant DNA carried on small, nonconjugative plasmids with broad host ranges can now be directly transferred to R. sphaeroides by this method.     

Conjugation in Rhodopseudomonas sphaeroides mediated by R plasmids

Plasmids R68.45, RP4, RP4::Mu cts62, RP1ts::Tn10, RP1ts::Tn9, Rts1 and RP41 were transferred into cells of photosynthetic nitrogen-fixation bacterium Rhodopseudomonas sphaeroides from Escherichia coli and Pseudomonas aeruginosa. The transfer of plasmids occurred with high frequency of 10(-1) to 10(-2) per donor cell in all cases. Mobilization of R. sphaeroides 2R chromosome was obtained by RP4 and Rts1 plasmids at a frequency of 10(-7) to 10(-8) per donor cell in all cases. Mobilization of R. sphaeroides 2R chromosome was obtained by RP4 and Rts1 plasmids at a frequency of 10(-7) to 10(-8) per donor cell. Bacteriophage Mu cts62 could be induced from the plasmid DNA in R. sphaeroides 2R cells and was capable of the lytic growth and producing phage progeny. It was demonstrated that an increase in the efficiency of donor chromosomal genes transfer into recipient cells could be achieved in crosses with the donor carrying RP4::Mcts62 plasmid.     

n-Alkane dissimilation by Rhodopseudomonas sphaeroides transferred OCT plasmid

The OCT plasmid from Pseudomonas maltophilia N246-1 was transferred to Rhodopseudomonas sphaeroides M1 with very low frequency (1.4–1.9 × 10^–5 per recipient cell at pH 7–8 for a 3-hour reaction time). P. maltophilia N246-1 was able to utilize C₈-C₁₄ of n-alkanes, whereas R. gas-liquid chromatography determined that the broad range of carbon numbers of n-alkanes in crude oil was remarkably degraded by the transconjugant, R. sphaeroides M1-C1, compared with donor strain N246-1. The fact that donor and transconjugant strains simultaneously lost the capacity to utilize n-alkanes on L-broth medium suggests that the OCT plasmids are unstable. It was found that the OCT plasmid of P. maltophilia N246 was incompatible with the IncP-2 group of P. aeruginosa KCTC 11245. Offprint requests to: K.-H. Min, Sookmyung Women's University.     

Intermembrane phospholipid transfer mediated by cell-free extracts of Rhodopseudomonas sphaeroides.

The transfer of phospholipids between two membrane substrates catalyzed by a soluble protein fraction from Rhodopseudomonas sphaeroides has been demonstrated. The assay employs purified intracytoplasmic membrane (ICM) vesicles derived from cells of R. sphaeroides grown on [3H]acetate as the phospholipid donor substrate and phosphatidylcholine (70%)/phosphatidylethanolamine (30%) unilamellar liposomes containing [14C]triolein, a nontransferable marker, as the acceptor substrate for transferred phospholipids. Incubation of these two membrane substrates with a 40 to 80% (NH4)2SO4 protein fraction from R. sphaeroides results in the transfer of tritium-labeled ICM phospholipids to the acceptor membrane substrate. Upon completion of the incubation period, the donor ICM vesicles are quantitatively separated from the acceptor liposomes by precipitation with antibody prepared against whole, purified ICM vesicles. Phospholipid transfer is linear with respect to time and protein concentration, is inhibited by trypsin and heat, and shows an absolute dependence upon the presence of acceptor liposomes and the 40 to 80% (NH4)2SO4 protein fraction. Control experiments indicate that no fusion of the donor and acceptor membrane occurs during the incubation period and that, following prolonged incubation there is no detectable degradation of the labeled lipid components. Preliminary data on the phospholipid specificity of the transfer reaction is also presented.     

R68.45 mediated chromosomal gene transfer in Agrobacterium tumefaciens

A large plasmid enables its host Agrobacterium tumefaciens to cause tumorous condition in a wide variety of dicotyledonous plants [see Ooms et al. Gene 14:33–50 (1981)]. The location and role of chromosomal genes in this phenomenon are not known. As the first stage in studying this aspect, a project was initiated to investigate the chromosomal genetics of the bacterium. R68.45, a P group plasmid, was chosen as a transmission agent. After a preliminary assessment it was decided to use C58 as a standard strain to carry out the mapping. The plasmid itself, as judged by the presence of antibiotic markers, appears to be stable in a. tumefaciens; its ability to promote chromosomal mobilisation, however, remains only in 60–80% transconjugants. Good Agrobacterium donors are capable of transferring chromosomal genes at a frequency varying between 10^-5 to 10^-6 per recipient. The recombinants are stable even under non-selective conditions. A linear linkage map consisting of 16 markers was built using coinheritance frequencies obtained from 21 four-point crosses.     

Glycerol dissimilation in Rhodopseudomonas sphaeroides.

Rhodopseudomonas sphaeroides followed a diauxic growth curve when grown on a malate-glycerol medium, the first phase of growth being supported by malate and the second by glycerol. A soluble glycerokinase and a particulate, pyridine nucleotide-independent glycerophosphate dehydrogenase, were induced by the presence of glycerol in the medium, but neither was fully expressed nor functional until all malate had been consumed.     

High-frequency chromosome transfer in Rhodopseudomonas sphaeroides promoted by broad-host-range plasmid RP1 carrying mercury transposon Tn501.

Insertion of the mercury resistance transposon Tn501 into broad-host-range plasmid RP1 greatly enhanced the ability of this plasmid to promote chromosome transfer in the photosynthetic bacterium Rhodopseudomonas sphaeroides. Compared with the wild-type RP1, which produced less than 10(-8) recombinants per donor cell, RP1::Tn501 produced between 10(-3) and 10(-7) recombinants per donor cell depending upon the marker selected. Plasmid RP1::Tn501 promoted polarized transfer of the chromosome from one or perhaps two origins on the chromosome, giving rise to two linkage groups. All of the biosynthetic and antibiotic resistance genes that have been mapped, including those involved in photosynthesis, occur on one or another of these linkage groups.     

Denitrification in Rhodopseudomonas sphaeroides f. denitrificans

Rhodopseudomonas sphaeroides f. denitrificans grown photosynthetically with NO ₃ ^- under anaerobic conditions accumulated NO ₂ ^- in the culture medium. In washed cells succinate, lactate, fumarate, citrate and malate, were effective electron donors for the reduction of NO ₃ ^- , NO ₂ ^- and N₂O to N₂ gas. Nitrate reductase was inhibited by amytal and potassium thocyanate. Nitrite reductase activity was severely restricted by potassium cyanide, sodium diethyldithiocarbamate, Amytal and 2-n-heptyl-4-hydroxyquinoline-N-oxide whereas N₂O reductase was inhibited by NaN₃, C₂H₂ and KCNS. Cells incubated with either K¹⁵NO₃ or K¹⁵NO₂ produced ¹⁵N₂O and ¹⁵N₂. A stoichiometry of 2:1 was recorded for the reduction of either NO ₃ ^- or NO ₂ ^- to N₂O and N₂ and for N₂O to N₂ it was 1:1.Abbreviations BVH reduced benzyl viologen - MVH reduced methyl viologen - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - CCCP carbonyl cyanide-m-chlorophenyl-hydrazone - DIECA diethyl dithiocarbamate - KCN potassium cyanide     

Nitrate reductase from Rhodopseudomonas sphaeroides.

The facultative phototroph Rhodopseudomonas sphaeroides DSM158 was incapable of either assimilating or dissimilating nitrate, although the organism could reduce it enzymatically to nitrite either anaerobically in the light or aerobically in the dark. Reduction of nitrate was mediated by a nitrate reductase bound to chromatophores that could be easily solubilized and functioned with chemically reduced viologens or photochemically reduced flavins as electron donors. The enzyme was solubilized, and some of its kinetic and molecular parameters were determined. It seemed to be nonadaptive, ammonia did not repress its synthesis, and its activity underwent a rapid decline when the cells entered the stationary growth phase. Studies with inhibitors and with metal antagonists indicated that molybdenum and possibly iron participate in the enzymatic reduction of nitrate. The conjectural significance of this nitrate reductase in phototrophic bacteria is discussed.     

Transfer of plasmids RP4 and R68.45 and chromosomal mobilization in cowpea rhizobia

R-plasmids RP4 and its derivatives R68.45 were transferred from Escherichia coli to two cowpea rhizobia strains. The frequency of RP4 transfer in cowpea rhizobia strains JRC23-SM20 and IRC256-HA409 was 1,000-fold higher than transfer frequency of R68.45. The transconjugants were further used to transfer R-plasmids within (isogenic) and between (non-isogenic) cowpea rhizobia strains. The plasmid transfer frequency was higher in isogenic than non-isogenic strains. The ability of R-plasmids to mobilize chromosomal genes in cowpea rhizobia was also examined. R-plasmids mediated the chromosomal transfer; however, mobilization of chromosomal markers Sm^R and Met⁺ by RP4 in isogenic strains was more efficient than by R68.45. Chromosomal mobilization has not previously been reported in cowpea rhizobia.Abbreviations Ap ampicillin - Km kanamycin - Tc tetracycline - Rif rifampicin - TYS tryptone yeast-extract sodium chloride - YEMA yeast-extract mannitol agar - YEMB yeast-extract mannitol broth Part of the work was presented in 6th International Symposium on Nitrogen Fixation at Oregon State University, Corvallis, August 4–10, 1985     

Rhodopseudomonas sphaeroides forma sp. denitrificans,a denitrifying strain as a subspecies of Rhodopseudomonas sphaeroides

From polluted water of a lagoon pond a new type of denitrifying photosynthetic purple bacteria was isolated. With respect to morphology, fine structure, photopigments, requirement for growth factors, the range of utilization of organic substrates for phototrophic growth and DNA base ratio, the denitrifying strains show the closest resemblance to Rhodopseudomonas sphaeroides and were therefore described as a subspecies named R. sphaeroides forma sp. denitrificans. The new isolates grow well with nitrate anaerobically in the dark accompanying the evolution of nitrogen gas. They cannot assimilate nitrate as the nitrogen source for growth.     

Mobilization of the genes for photosynthesis from Rhodopseudomonas capsulata by a promiscuous plasmid.

Plasmid pBLM2, a derivative of RP1 with enhanced chromosome mobilization activity in Rhodopseudomonas capsulata, was isolated by screening rare exconjugant clones for sex factor activity. pBLM2 mobilized all known genes affecting photosynthesis as well as chromosomal genes for streptomycin and rifampin resistance and tryptophan and cytochrome biosynthesis. Tight linkage was exhibited among the genes affecting photosynthesis. The frequency of successful transfer of chromosomal markers reached 6 X 10(-4) per donor cell. R-primes were occasionally formed during conjugation, and a number of R-primes bearing the genes for photosynthesis were isolated by screening R. capsulata exconjugants with complementation phenotypes for the ability to transmit plasmid-borne R. capsulata genes to Escherichia coli cells. These R-primes were unstable in R. capsulata, but stable in E. coli or Pseudomonas fluorescens. Complementation and recombination events that occurred upon introduction of R-primes into R. capsulata mutants with altered photosynthetic apparatuses could be visualized as variations in colony pigmentation. Each R-prime studied complemented all known types of mutation affecting the differentiation of the photosynthetic apparatus, and no other R. capsulata gene was identified on those plasmids. The R. capsulata genes borne on the R-primes were not functional in E. coli or P. fluorescens.     

Ferric iron reductase of Rhodopseudomonas sphaeroides.

Partially digested chromosomal DNA of Bacillus brevis ATCC 9999, a producer of the cyclic peptide antibiotic gramicidin S, was ligated into the BamHI site of the Escherichia coli expression vector pUR2-Bam. The ligated molecules were used to transfer E. coli to ampicillin resistance. Of 5 X 10(3) colonies tested by in situ immunoassay for a cross-reaction with antibodies against the gramicidin S synthetase 2, 6 colonies were found to be immunoreactive. A clone designated MK2, which had a 3.9-kilobase insert of B. brevis DNA, directed in E. coli under the lac promoter control the synthesis of polypeptides that were cross-reactive with the antibody to the gramicidin S synthetase 2. Partial purification of the gene products by gel filtration revealed a major fraction with an approximate molecular weight of 140,000 and with specific ornithine-dependent ATP-32PPi and 2'-dATP-32PPi exchange activities. These unique activities of the gramicidin S synthetase 2 were not detected in the E. coli strain harboring the vector.     

Light-induced, carrier-mediated transport of tetracycline by Rhodopseudomonas sphaeroides.

Tetracycline accumulation by the phototrophic bacterium Rhodopseudomonas sphaeroides has been studied, using the fluorescence properties of the antibiotic and measuring uptake of [7- 3H]tetracycline. Accumulation was carrier mediated, with a Km of approximately 300 micronM. Efflux also appeared to be carried mediated, with a Km of 25 mM. Chlorotetracycline competitively inhibited tetracycline transport. The transport was energy dependent. Efflux occurred during the influx process, and an energy-requiring steady state was reached when influx balanced efflux. Transport was inhibited by metabolic inhibitors such as antimycin A, cyanide, and iodoacetate. Proton conductors such as carbonylcyanide m-chlorophenyl hydrazone were strongly inhibitory. Efflux was not energy dependent. Efflux is partially blocked by mercuric ions and completely blocked by an external pH of 9 to 11. Although efflux rates increased continuously with lowering of the pH, influx rates have a sharp maximum at pH 7.     

Plasmid transfer and expression in Rhodopseudomonas sphaeroides.

Plasmid RP4 (among others) has been transferred from Escherichia coli to Rhodopseudomonas sphaeroides. Data bearing on the physical presence of the plasmid and its expression of drug resistance determinants in R. sphaeroides are presented. Conditions of transfer between R. sphaeroides strains, between R. sphaeroides and R. capsulata, and between R. sphaeroides and E. coli have been carefully defined.     

Protein composition of Rhodopseudomonas sphaeroides outer membrane.

The outer membrane polypeptide profile of Rhodopseudmonas sphaeroides was characterized. Solubilization of the outer membrane at 75 or 100 degrees C as opposed to room temperature resulted in the dissociation of 75-, 72-, and 68-kilodalton (kdal) polypeptide aggregates into 29-, 26.5-, and 21.5-kdal polypeptides, respectively, and a shared 47-kdal subunit. Similarly, an 88.5-kdal polypeptide dissociates into a 45-kdal monomeric form, and the electrophoretic mobility of a 58.5-kdal polypeptide was altered to 83 kdal.Lysozyme treatment of outer membrane fractions altered the 21.5-kdal polypeptide mobility to 23 kdal. The presence of lipid in both the 47-kdal polypeptide and an 8- to 10-kdal polypeptide was demonstrated by lipid staining and [14C]acetate incorporation. The lipid component of the 47-kdal polypeptide was neither lipopolysaccharide nor phospholipid. The 8- to 10-kdal polypeptide may be the equivalent of the Braun lipoprotein. Outer membrane fractions isolated from R. sphaeroides-specific phage RS1-resistant mutants were deficient in several of the high-molecular-weight aggregates involving the 47-kdal polypeptide.