Glutathione-dependent formaldehyde dehydrogenases (GSH-FDH) represent a ubiquitous class of enzymes, found in both prokaryotes and eukaryotes. During the course of studying energy-generating pathways in the photosynthetic bacterium Rhodobacter sphaeroides, a gene (adhI) encoding a GSH-FDH homolog has been identified as part of an operon (adhI-cycI) that also encodes an isoform of the cytochrome c 2 family of electron transport proteins (isocytochrome c 2 ). Enzyme assays with crude Escherichia coli extracts expressing AdhI show that this protein has the characteristic substrate preference of a GSH-FDH. Ferguson plot analysis with zymograms suggests that the functional form of AdhI is a homodimer of ϳ40-kDa subunits, analogous to other GSH-FDH enzymes. These properties of AdhI were used to show that mutations which increase or decrease adhI expression change the specific activity of GSH-FDH in R. sphaeroides extracts. In addition, expression of the presumed adhI-cycI operon appears to be transcriptionally regulated, since the abundance of the major adhI-specific primer extension product is increased by the trans-acting spd-7 mutation, which increases the level of both isocytochrome c 2 and AdhI activity. While transcriptional linkage of adhI and cycI could suggest a function in a common metabolic pathway, isocytochrome c 2 (periplasm) and AdhI (cytoplasm) are localized in separate compartments of R. sphaeroides. Potential roles for AdhI in carbon and energy generation and the possible relationship of GSH-FDH activity to isocytochrome c 2 will be discussed based on the commonly accepted physiological functions of GSH-FDH enzymes in prokaryotes and eukaryotes.Most organisms have the ability, by using various metabolic pathways, to generate both carbon and energy from the oxidation of a wide spectrum of substrates. The function of these metabolic pathways often requires the coordinated expression of specific enzymes for substrate oxidation and novel electron carriers to generate cellular energy from this substrate. To understand how different metabolic pathways provide cells with both carbon and energy, we are studying the regulation and function of energy-generating systems in the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides.While energy-generating pathways in R. sphaeroides have been extremely well analyzed from biochemical and bioenergetic standpoints (24), genetic analysis has uncovered the existence of unexpected electron transport proteins. For example, recent experiments have identified a c-type cytochrome, isocytochrome c 2 , which allows R. sphaeroides Spd mutants to grow photosynthetically in the absence of cytochrome c 2 , the normal electron donor to the light-oxidized reaction center complexes (29). While the function of isocytochrome c 2 as a photosynthetic electron carrier in Spd mutants has been confirmed both genetically (31) and biochemically (46), a physiological role for this protein in wild-type cells is not known. Because mutational and complementation analysis indicated th...

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...

In Rhodobacter sphaeroides, cytochrome c2 (cyt c2) is a periplasmic redox protein required for photosynthetic electron transfer. cyt c2-deficient mutants created by replacing the gene encoding the apoprotein for cyt c2 (cycA) with a kanamycin resistance cartridge are photosynthetically incompetent. Spontaneous mutations that suppress this photosynthesis deficiency (spd mutants) arise at a frequency of 1 to 10 in 107. We analyzed the cytochrome content of several spd mutants spectroscopically and by heme peroxidase assays. These suppressors lacked detectable cyt c2, but they contained a new soluble cytochrome which was designated isocytochrome c2 (isocyt c2) that was not detectable in either cycA+ or cycA mutant cells. When spd mutants were grown photosynthetically, isocyt c2 was present at approximately 20 to 40% of the level of cyt c2 found in photosynthetically grown wild type cells, and it was found in the periplasm with cytochromes c' and C554. These spd mutants also had several other pleiotropic phenotypes. Although photosynthetic growth rates of the spd mutants were comparable to those of wild-type strains at all light intensities tested, they contained elevated levels of B800-850 pigment-protein complexes. Several spd mutants contained detectable amounts of isocyt c2 under aerobic conditions. Finally, heme peroxidase assays indicated that, under anaerobic conditions, the spd mutants may contain another new cytochrome in addition to isocyt c2. These pleiotropic phenotypes, the frequency at which the spd mutants arise, and the fact that a frameshift mutagen is very effective in generating the spd phenotype suggest that some spd mutants contain a mutation in loci which regulate cytochrome synthesis. Rhodobacter sphaeroides is

An antimicrobial phenolic stilbene, (E)-3-hydroxy-5-methoxystilbene, 1 was recently isolated from the leaves of Comptonia peregrina (L.) Coulter and shown to possess inhibitory activity against several Gram-positive bacteria, including isolates of methicillin-resistant Staphylococcus aureus (MRSA), Mycobacterium bovis BCG, and avirulent Bacillusanthracis (Sterne strain), among others. These results prompted the design and synthesis of two new classes of compounds, phenoxystyrenes and phenothiostyrenes, as analogs of the natural antimicrobial stilbene. These and additional stilbenoid analogs were synthesized using new, efficient, copper-mediated coupling strategies. Minimum inhibitory concentration (MIC) antimicrobial assays were performed on all compounds prepared. These preliminary structure-activity relationship studies indicated that both new classes of synthetic analogs, as well as the stilbenes, show promising activity against Gram-positive bacteria when at least one phenolic moiety is present, but not when absent. The potencies of the phenolic phenoxystyrenes and phenothiostyrenes were found to be comparable to those of the phenolic stilbenes tested.

Antibacterial bioassay-guided fractionation of two American mushroom species, Jahnoporus hirtus and Albatrellus flettii, led to the isolation and identification of their major antibacterial constituents: 3,11-dioxolanosta-8,24( Z)-diene-26-oic acid (1) from J. hirtus and confluentin (2), grifolin (3), and neogrifolin (4) from A. flettii. Compound 1 is a new lanostane-type triterpene. All purified compounds were evaluated for their ability to inhibit the growth of Bacillus cereus and Enterococcus faecalis using standard MIC assays. Compounds 1- 4 demonstrated MIC values of 40, 20, 10, and 20 microg/mL, respectively, against B. cereus and MIC values of 32, 1.0, 0.5, and 0.5 microg/mL, respectively, against E. faecalis. Thus, one novel compound and three others were shown to possess antimicrobial activities against these gram-positive bacteria employed as surrogates for more virulent and dangerous pathogens.