In Rhodobacter sphaeroides, cytochrome c2 (cyt c2)-deficient mutants are photosynthetically incompetent (PS-). However, mutations which suppress the photosynthetic deficiency (spd mutations) of cyt c2 mutants increase the levels of a cyt c2 isoform, isocyt c2. To determine whether isocyt c2 was required for photosynthetic growth of Spd mutants, we used TnS mutagenesis to generate a PS-mutant (TP39) that lacks both cyt c2 and isocyt c2. DNA sequence analysis of wild-type DNA that restores isocyt c2 production and photosynthetic growth to TP39 indicates that it encodes the isocyt c2 structural gene, cycL. The Tn5 insertion in TP39 is -1.5 kb upstream of cycI, and our results show that it is polar onto cycL. The cycI gene has been physically mapped to a region of chromosome I that is -700 kb from the R. sphaeroides photosynthetic gene cluster. Construction of a defined cycI null mutant and complementation of several mutants with the cycI gene under the control of the cyt c2 promoter region indicate that an increase in the levels of isocyt c2 alone is necessary and sufficient for photosynthetic growth in the absence of cyt c2. The data are discussed in terms of the obligate role of isocyt c2 in cyt c2-independent photosynthesis of R. sphaeroides. sphaeroides DNA (10). The concentration of tetracycline used with pRK415-derived plasmids (15) was increased to 10 ,ug/ml for E. coli only. Trimethoprim, suspended in dimethylformamide, was used at 30 ,g/ml for R. sphaeroides and 100 ,ug/ml for E. coli. Spectinomycin was used at 25 ,g/ml for both R. sphaeroides and E. coli. For maintenance of pUC-derived plasmids, ampicillin was added to E. coli cultures at 50 ,ug/ml. R sphaeroides was grown in Sistrom's minimal medium A (16, 31) as previously described (8). Cell growth was measured turbidimetrically (35), and cultures were harvested during the exponential phase (6 x 108 to 1.5 x 109 cells per ml) (25). For photosynthetic growth on solid media, GasPak (BBL Microbiology Systems) jars with H2-CO2 generators were used.Transposon mutagenesis with pSUPTP5. Because the strain used for TnS mutagenesis contained a genomic kan gene (25), the use of wild-type TnS was not practical. Thus, a TnS Tpr derivative (28) was cloned as an -5.5-kb BamHI fragment into pSUP202 (30) to generate pSUPTP5. Control experiments showed that this derivative transposed with approximately the same frequency as wild-type TnS and that a spectrum of auxotrophic and pigment mutants could be obtained (lla).To isolate an isocyt c2 mutant, pSUPTP5 was mobilized into R sphaeroides CYCA65R7 (25) to generate independent transposition events. Briefly, -40 ml of an exponentialphase CYCA65R7 culture was harvested by centrifugation, and on top of this, -40 ml of a saturated S17-1(pSUPTP5) culture was collected. The cells were gently suspended in 4 ml of Sistrom's medium, and 120 RI was spread onto a series of LB plates. After incubation for 3 h at 32°C, the cells were replica plated to Sistrom's medium containing trimethoprim and incubated under aerobic conditions. Fro...

Abstract. The differentiation of homologous chromosomes as well as their parental origin can presently be conducted and determined exclusively by molecular genetic methods using microsatellite or SNP analysis. Only in exceptional cases is a distinction on a single-cell level possible, e.g. due to variations within the heterochromatic regions of chromosomes 1, 9, 16 and Y or the p-arms of the acrocentric chromosomes. In the absence of such polymorphisms, an individual distinction of the homologous chromosomes is not currently possible. Consequently, various questions of scientific and diagnostic relevance are unable to be answered. Based on the recently detected large-scale copy-number variations (LCV) or copynumber polymorphisms (CNP) spanning up to several megabase pairs of DNA, in this study, a molecular cytogenetic technique for the inter-individual differentiation of homologous chromosomes called parental-origin-determination fluorescence in situ hybridization (pod-FISH) is presented. All human chromosomes were covered with 225 LCVand/or CNP-specific BAC probes, and one-to five-color chromosome-specific pod-FISH sets were created, evaluated and optimized. We demonstrated that pod-FISH is suitable for single-cell analysis of uniparental disomy (UDP) in clinical cases such as Prader-Willi syndrome caused by maternal UPD. A rare clinical case with a mosaic form of a genome-wide isodisomy was used to determine the detection limits of pod-FISH. Additionally we analyzed the informativeness of conventional microsatellite analysis for the first time and compared the results to pod-FISH. With this new possibility to study the parental origin of individual human chromosomes on a single-cell level, new doors for diagnostic and basic research are opened.

A glutathione (GSH)-dependent pathway is used for formaldehyde metabolism by a wide variety of prokaryotes and eukaryotes. In this pathway, S-hydroxymethylglutathione, produced by the reaction of formaldehyde with the thiolate moiety of glutathione, is the substrate for a GSH-dependent formaldehyde dehydrogenase (GSH-FDH). While expression of GSH-FDH often increases in the presence of metabolic or exogenous sources of formaldehyde, little is known about the factors that regulate this response. Here, we identify two signal transduction pathways that regulate expression of adhI, the gene encoding GSH-FDH, in Rhodobacter sphaeroides. The loss of the histidine kinase response regulator pair RfdRS or the histidine kinase RfdS increases adhI transcription in the absence of metabolic sources of formaldehyde. Cells lacking RfdRS further increase adhI expression in the presence of metabolic sources of formaldehyde (methanol), suggesting that this negative regulator of GSH-FDH expression does not respond to this compound. In contrast, mutants lacking the histidine kinase response regulator pair AfdRS or the histidine kinase AfdS cannot induce adhI expression in the presence of either formaldehyde or metabolic sources of this compound. AfdR stimulates activity of the adhI promoter in vitro, indicating that this protein is a direct activator of GSH-FDH expression. Activation by AfdR is detectable only after incubation of the protein with acetyl phosphate, suggesting that phosphorylation is necessary for transcription activation. Activation of adhI transcription by acetyl-phosphate-treated AfdR in vitro is inhibited by a truncated RfdR protein, suggesting that this protein is a direct repressor of GSH-FDH expression. Together, the data indicate that AfdRS and RfdRS positively and negatively regulate adhI transcription in response to different signals.Formaldehyde is a cytogenic compound that is produced by environmental, industrial, and metabolic processes including the oxidative demethylation of amino acids and osmoprotectants or oxidation of one-carbon compounds like methanol, methyl halides, and methane (10, 11, 16, 20-22, 24, 35, 37, 38). The toxicity of formaldehyde stems from its reactivity with amino and sulfhydryl groups of biological molecules, causing alkylation, mutations, and cross-links that destroy the function of membranes, proteins, and nucleic acids (18,25). When the sources of formaldehyde, its toxicity, and the carbon skeletons and reducing power derived from its oxidation are considered, it is clear that cells benefit from metabolism of this compound.We are studying formaldehyde metabolism by the facultative bacterium Rhodobacter sphaeroides. This ␣-proteobacterium uses a glutathione (GSH)-dependent pathway for formaldehyde metabolism that is similar to those present in several prokaryotic and eukaryotic cells (5, 7). In this pathway, formaldehyde reacts with the thiolate moiety of GSH to form Shydroxymethylglutathione, a substrate for a GSH-dependent formaldehyde dehydrogenase (GSH-FDH). GSH-FDH oxidizes...

Rhodobacter sphaeroides strains lacking cytochrome c2 (cyt c2), the normal electron donor to P870+ in light-oxidized reaction center (RC) complexes, are unable to grow photosynthetically. However, spd mutations that suppress the photosynthetic deficiency of cyt c2 mutants elevate levels of the cyt c2 isoform, isocyt c2. We monitored photosynthetic electron transfer in whole cells, in chromatophores, and with purified components to ascertain if and how isocyt c2 reduced light-oxidized RC complexes. These studies revealed that several fundamental aspects of photosynthetic electron transfer were similar in strains that use isocyt c2 and wild-type cells. For example, P870+ reduction accompanied cytochrome c oxidation. In addition, photosynthetic electron transfer was blocked by the well-known cyt bc1 complex inhibitors antimycin and myxothiazol. However, even at the increased isocyt c2 levels present in these strains (approximately 40% that of cyt c2 in wild-type cells), there was little, if any, of the rapid (< 5 microns) electron transfer to P870+ that is characteristic of cytochromes bound to RC complexes at the time of the light flash. Thus, it appears that isocyt c2 function limits the in vivo rate of P870+ reduction. Indeed, at low ionic strength in vitro, the apparent affinity of isocyt c2 for RC complexes (KD approximately 40 microM) is significantly lower than that of cyt c2 (KD approximately 1.0 microM). This reduced affinity does not appear to result from an altered mode of RC binding by isocyt c2 since electrostatic interactions make similar overall contributions to the binding of both cyt c2 and isocyt c2 to this membrane-bound redox partner. Thus, sequence, structural, or local conformational differences between cyt c2 and isocyt c2 significantly alter their apparent affinities for this physiologically relevant redox partner.