Raymond P. Cox

Microbial production of skatole in the hind gut of pigs given different diets and its relation to skatole deposition in backfat

The intestinal production of skatole and its deposition in backfat was investigated in 35 uncastrated crossbred male pigs. The pigs were fed five purified non-commercial diets containing either casein or brewers yeast slurry as protein source. The yeast slurry diet was used alone or supplemented with either wheat bran (200 g/kg), sugar-beet pulp (200 g/kg), or soya oil (100 g/kg). Skatole concentrations in backfat, and in digesta in different sections of the gastro-intestinal tract were measured at slaughter (mean weight 112 kg). There were large variations in skatole concentrations in the hind gut of different animals given the same diet. Although there was some correlation between skatole in intestinal contents and deposition in adipose tissue, there were also large variations between individual animals in their response to intestinal skatole. Nevertheless, there was a clear effect of diet on both intestinal skatole production and skatole deposition in backfat. The use of casein as a protein source decreased microbial skatole production, the total amount in the gut, and the concentration in the backfat. Addition of sugar-beet pulp to the yeast slurry diet increased microbial activity in the intestine (measured as ATP content, concentration of short-chain fatty acids, and lowering of digesta pH ). There was a decreased rate of skatole production during in vitro incubations of intestinal content, and less skatole in the hind gut and backfat. In vitro fermentations of freeze-dried Heal effluent inoculated with faecal bacteria, and addition of substrates to in vitro incubations of intestinal contents, demonstrated that tryptophan availability rather than microbial activity was the limiting factor for skatole production. The results show that skatole production depends on the amount of protein entering the hind gut and the proteolytic activity of the intestinal microbiota. Protein fermentation in the hind gut can be decreased either by using more readily digestible protein sources (for example casein rather than yeast slurry) which reduce the amount of protein passing through to the hind gut, or by adding an alternative energy source which is more readily metabolized by the hind gut microbiota (for example supplementation of the yeast slurry diet with sugar-beet pulp). This provides a basis for the rational design of diets which will decrease skatole concentrations in the carcass.

Lateral and transverse organisation of cytochromes in the chloroplast thylakoid membrane

Abstract We have measured the cytochrome compositions of subfractions derived from appressed and non-appressed thylakoids by centrifugation and aqueous two-phase partition. Cytochrome b -559 (HP) was not detectable in the fraction derived from non-appressed thylakoids. Cytochromes f , b -563 and b -559 (LP) were all evenly distributed throughout the thylakoid membrane. This distribution points to plastocyanin as a possible lateral shuttle of reducing equivalents between spatially separated photosystems. Cytochrome f was accessible to externally added plastocyanin in the inside-out vesicles but not in vesicles of normal sidedness. This strongly supports a location at the inner side of the thylakoid membrane. Cytochrome b -563 was slowly reduced by dithionite in vesicles with both normal and inside-out orientation suggesting a location within the membrane interior.

Fluorescence polarization and spin-label studies of the fluidity of stromal and granal chloroplast membranes

Abstract The lipid fluidity of thylakoid membrane regions separated by Yeda press and sonication methods has been investigated using diphenylhexatriene fluorescence polarization measurements and rotational correlation times derived from the ESR spectra of the spin-labels 5-doxyldecane and 12-doxylstearate. According to both techniques, stromal lamellae vesicles with essentially only Photosystem I activity were more fluid than the granal membranes. The differences in lipid fluidity between the two fractions were interpreted in terms of the ratio of the amounts of protein compared to lipid in the membranes. Stromal lamellae fractions contained lower protein/lipid ratios compared with the granal membranes.

LIGHT INTENSITY EFFECTS ON PIGMENT COMPOSITION AND ORGANISATION IN THE GREEN SULFUR BACTERIUM CHLOROBIUM TEPIDUM

We have investigated the changes in the pigment composition and organisation of the light-harvesting apparatus of the green sulfur bacterium Chlorobium tepidum growing under different light intensities. Cells grown at lower light intensities had lower exponential growth rates and increased amounts of the main light-harvesting pigments, bacteriochlorophyll c and carotenoids, on a cell protein basis. Absorption spectra of chlorosomes isolated from cells grown at low light intensities revealed a red-shift of up to 8 nm in the Qy band of bacteriochlorophyll c compared to chlorosomes from high light grown cells. A similar red-shift of up to 4 nm was also observed in the corresponding fluorescence emission peaks. HPLC analysis of pigment extracts showed a correlation between the red-shift and the content of the more alkylated BChl c homologs, which increased as light intensity for growth was lower. Furthermore, analysis of the carotenoid composition in chlorosomes re vealed a conspicuous change in the ratio between chlorobactene and 1′, 2′-dihydrochlorobactene, which dramatically decreased from 5 to 0.7 in light-limited cultures.

Studies of the location and function of isoprenoid quinones in chlorosomes from green sulfur bacteria

The chlorosome antenna of the green sulfur bacterium Chlorobium tepidum essentially consists of aggregated bacteriochlorophyll (BChl) c enveloped in a glycolipid monolayer. Small amounts of protein and the isoprenoid quinones chlorobiumquinone (CK) and menaquinone-7 (MK-7) are also present. Treatment of isolated chlorosomes from Cb. tepidum with sodium dodecyl sulfate (SDS) did not affect the quinones, demonstrating that these are located in a site which is inaccessible to SDS, probably in the interior of the chlorosomes. About half of the quinones were removed by Triton X-100. The non-ionic character of Triton probably allowed it to extract components from within the chlorosomes. MK-10 in chlorosomes from the green filamentous bacterium Chloroflexus aurantiacus was likewise found to be located in the chlorosome interior. The excitation transfer in isolated chlorosomes from Cb. tepidum is redox-regulated. We found a ratio of BChl c fluorescenceintensity under reducing conditions (Fred) to that under oxidizing conditions (Fox) of approximately 40. The chlorosomal BChl a fluorescence was also redox-regulated. When the chlorosomal BChl c–BChl c interactions were disrupted by 1-hexanol, the BChl c Fred/Fox ratiodecreased to approximately 3. When CK and MK-7 were extracted from isolated chlorosomes with hexane, the BChl c Fred/Fox ratio also decreased to approximately 3. A BChl c Fred/Fox ratio of 3–5 was furthermore observed in aggregates of pure BChl c and in chlorosomes from Cfx. aurantiacus which do not contain CK. We therefore suggest that BChl c aggregates inherently exhibit a small redox-dependent fluorescence (Fred/Fox ≈ 3) and that the large redox-dependent fluorescence observed in chlorobial chlorosomes (Fred/Fox ≈ 40) is CK-dependent.

Changes in Bacteriochlorophyll c Organization during Acid Treatment of Chlorosomes from Chlorobium tepidum

Abstract— We have investigated changes in the organization of bacteriochlorophyll c (BChl c) in chlorosomes isolated from the green sulfur bacterium Chlorobium tepidum during the formation of bacteriopheophytin and bacteriopheo‐phorbide in acidic media. The reaction was much slower than that observed with BChl dissolved in methanol, suggesting that the aggregation of BChl or the presence of the chlorosome envelope constitutes a barrier to the reaction with protons in the aqueous phase. In most cases the first two‐thirds of the reaction showed time courses that were close to linear. Simultaneously with the pheo‐phytinization process we observed a red‐shift of the Qy band of the remaining aggregated BChl c reaching a maximum extent of 9 nm. Analysis of the spectral changes provided evidence at least for two spectrally distinct pools of aggregated BChl c with different rates of reaction with acid. An HPLC analysis showed that there were no changes in the distribution of the four major homologs of BChl c remaining in chlorosomes during the course of acid treatment, at least up to the time when two‐thirds had been converted to pheophytin. This suggests that the homologs of BChl c are uniformly distributed within the chlorosome.

Effect of oxygen concentration on dark nitrogen fixation and respiration in cyanobacteria

Simultaneous measurements of acetylene reduction by Anabaena variabilis and the concentration of dissolved oxygen in the suspension were made using a specially designed vessel which allowed measurements under steady-state conditions. The rate of acetylene reduction in the dark increased with increasing oxygen concentrations until a maximum value was reached at 300 μM O2 (corresponding to 30% O2 in the gas phase at 35°C). This presumably results from a requirement for energy provided by respiration. Measurements of the dependence of respiration rate on dissolved oxygen concentration were made under comparable conditions using an open system to allow conditions close to steady-state to be obtained. The respiration rate of diazotrophically grown Anabaena variabilis had a dependence on oxygen concentration corresponding to the sum of two activities. These had Km values of 1.0 μM and 69 μM and values of Vmax of similar magnitude. Only the high affinity activity was observed in nitrate-grown cyanobacteria lacking heterocysts, and this presumably represent activity in the vegetative cells. The oxygen concentration dependence of the low affinity activity resembled that for the stimulation of acetylene reduction. We interpret this as the result of oxygen uptake by the heterocysts. The results are consistent with the idea that in intact filaments of cyanobacteria O2 enters heterocysts much more slowly than it enters the vegetative cells.

Manipulation of the bacteriochlorophyll c homolog distribution in the green sulfur bacterium Chlorobium tepidum

We have shown that the green sulfur bacterium Chlorobium tepidum can be grown in batch culture supplemented with potentially toxic fatty alcohols without a major effect on the growth rate if the concentration of the alcohols is kept low either by programmed addition or by adding the alcohol as an inclusion complex with β-cyclodextrin. HPLC and GC analysis of pigment extracts from the supplemented cells showed that the fatty alcohols were incorporated into bacteriochlorophyll c as the esterifying alcohol. It was possible to change up to 43% of the naturally occurring farnesyl ester of bacteriochlorophyll c with the added alcohol. This change in the homolog composition had no effect on the spectral properties of the cells when farnesol was partially replaced by stearol, phytol or geranylgeraniol. However, with dodecanol we obtained a blue-shift of 6 nm of the Qy band of the bacteriochlorophyll c and a concomitant change in the fluorescence emission was observed. The possible significance of these findings is discussed in the light of current ideas about bacteriochlorophyll organization in the chlorosomes.

Incorporation of exogenous long-chain alcohols into bacteriochlorophyll c homologs by Chloroflexus aurantiacus

Chloroflexus aurantiacus grown in batch culture took up exogenous alcohols and incorporated these into bacteriochlorophyll c as the esterifying alcohol. It was possible to change the distribution of the naturally occurring homologs of bacteriochlorophyll c esterified with phytol, hexadecanol, and octadecanol by adding the appropriate alcohol. The corresponding homolog then made up at least 60% of the cellular bacteriochlorophyll c. It was also possible to obtain novel bacteriochlorophyll homologs not found in detectable amounts in control cells by adding fatty alcohols with short chains (C10, C12) or long chains (C20). These changes in bacteriochlorophyll composition had no detectable effects on the spectral properties of the chlorosomes.

BACTERIOCHLOROPHYLL ORGANIZATION AND ENERGY TRANSFER KINETICS IN CHLOROSOMES FROM CHLOROFLEXUS AURANTIACUS DEPEND ON THE LIGHT REGIME DURING GROWTH

We have used measurements of fluorescence and circular dichroism (CD) to compare chlorosome-membrane preparations derived from the green filamentous bacterium Chloroflexus aurantiacus grown in continuous culture at two different light-intensities. The cells grown under low light (6 μmol m−2 s−1) had a higher ratio of bacteriochlorophyll (BChl) c to BChl a than cells grown at a tenfold higher light intensity; the high-light-grown cells had much more carotenoid per bacteriochlorophyll.The anisotropy of the QY band of BChl c was calculated from steady-state fluorescence excitation and emission spectra with polarized light. The results showed that the BChl c in the chlorosomes derived from cells grown under high light has a higher structural order than BChl c in chlorosomes from low-light-grown cells. In the central part of the BChl c fluorescence emission band, the average angles between the transition dipole moments for BChl c molecules and the symmetry axis of the chlorosome rod element were estimated as 25° and 17° in chlorosomes obtained from the low- and high-light-grown cells, respectively.This difference in BChl organization was confirmed by the decay associated spectra of the two samples obtained using picosecond single-photon-counting experiments and global analysis of the fluorescence decays. The shortest decay component obtained, which probably represents energy-transfer from the chlorosome bacteriochlorophylls to the BChl a in the baseplate, was 15 ps in the chlorosomes from high-light-grown cell but only 7 ps in the preparation from low-light grown cells. The CD spectra of the two preparations were very different: chlorosomes from low-light-grown cells had a type II spectrum, while those from high-light-grown cells was of type I (Griebenow et al. (1991) Biochim Biophys Acta 1058: 194–202). The different shapes of the CD spectra confirm the existence of a qualitatively different organization of the BChl c in the two types of chlorosome.