Phototrophic growth and chlorosome formation in Chloroflexus aurantiacus under conditions of carotenoid deficiency

Chloroflexus aurantiacus was grown phototrophically in the presence of 60 µg of 2-hydroxybiphenyl (HBP) per ml, which inhibited the formation of colored carotenoids up to about 90%, while bacteriochlorophyll (BChl) c formation and phototrophic growth were not significantly influenced. When the HBP concentration was increased, the latter two processes became inhibited, yet, no further inhibition of carotenoid formation was possible. The typical shape of chlorosomes was not affected after growth in the presence of 60 µg of HBP per ml. However, the chlorosome dimensions were slightly decreased. Chlorosome preparations from carotenoid-deficient cultures lacked significant amounts of colored carotenoids and exhibited lower buoyant densities than chlorosomes from uninhibited control cultures. As compared on a BChl c basis, the relative contents of the two larger chlorosomal polypeptides of 10.8 and 15.5 kDa (apparent M_r= 11000 and 18000) were not affected by HBP, while formation of the 5.7 kDa (M_r= 3700) polypeptide was inhibited by 50%. The data suggest that, in C. aurantiacus, colored carotenoids are largely dispensable for phototrophic growth and chlorosome formation. Moreover, chlorosome formation and BChl c incorporation into chlorosomes in the absence of about half of the regular amount of the 5.7 kDa polypeptide excludes a constant relationship between these parameters.     

The organization of bacteriochlorophyll c in chlorosomes from Chloroflexus aurantiacus and the structural role of carotenoids and protein

The organization of bacteriochlorophyll c (BChl c) molecules was studied in normal and carotenoid-deficient chlorosomes isolated from the green phototrophic bacterium Chloroflexus aurantiacus. Carotenoid-deficient chlorosomes were obtained from cells grown in the presence of 60 µg of 2-hydroxybiphenyl per ml. At this concentration, BChl c synthesis was not affected while the formation of the 5.7 kDa chlorosome polypeptide was inhibited by about 50% (M. Foidl et al., submitted). Absorption, linear dichroism and circular dichroism spectroscopy showed that the organization of BChl c molecules with respect to each other as well as to the long axis of the chlorosomes was similar for both types of chlorosomes. Therefore, it is concluded that the organization of BChl c molecules is largely independent on the presence of the bulk of carotenoids as well as of at least half of the normal amount of the 5.7 kDa polypeptide. The Stark spectra of the chlorosomes, as characterized by a large difference polarizability for the ground- and excited states of the interacting BChl c molecules, were much more intense than those of individual pigments. It is proposed that this is caused by the strong overlap of BChl c molecules in the chlorosomes. In contrast to individual chlorophylls, BChl c in chlorosomes did not give rise to a significant difference permanent dipole moment for the ground- and excited states. This observation favors models for the BChl c organization which invoke the anti-parallel stacking of linear BChl c aggregates above those models in which linear BChl c aggregates are stacked in a parallel fashion. The difference between the Stark spectrum of carotenoid-deficient and WT chlorosomes indicates that the carotenoids are in the vicinity of the BChls.     

Hydrolysis of isolated coffee mannan and coffee extract by mannanases of Sclerotium rolfsii

Different mannanase preparations obtained from the filamentous fungus Sclerotium rolfsii were used for the hydrolysis of coffee mannan, thus reducing significantly the viscosity of coffee extracts. Mannan is the main polysaccharide component of these extracts and is responsible for their high viscosity, which negatively affects the technological processing of instant coffee. Coffee mannan was isolated from green defatted Arabica beans by delignification, acid wash and subsequent alkali extraction with a yield of 12.8%. Additionally, coffee extract polysaccharides were separated by alcohol precipitation and were found to form nearly half of the coffee extract dry weight. These isolated mannans as well as the mannan in the coffee extract were efficiently hydrolysed by the S. rolfsii mannanase, which resulted in significant viscosity reductions. Concurrently, the reducing sugar content increased continuously due to the release of various mannooligosaccharides including mannotetraose, mannotriose, and mannobiose. Both a partially purified, immobilised and a soluble, crude mannanase preparation were successfully employed for the degradation of coffee mannan.     

Bacteriochlorophyllc formation and chlorosome development inChloroflexus aurantiacus

The dependence of chlorosome development on bacteriochlorophyll (BChl)c synthesis was studied with the phototrophic green bacteriumChloroflexus aurantiacus. By selecting defined culture conditions, three possibilities could be identified. Upon addition of 5-aminolevulinic acid, cells of resting cultures increased their specific BChlc contents as well as the volumes of already existing chlorosomes. The number of chlorosomes, however, remained constant. Serine-limited chemostat cultures grown under steady state conditions exhibited constant rates of synthesis of both BChlc as well as of chlorosomes. The volume of the latter remained constant, as well. Upon addition of ALA to chemostat cultures, chlorosomes were synthesized at the same rate as before but their volumes increased as a consequence of increased BChlc incorporation. In chlorosomes isolated from resting cultures supplied with ALA the amounts of all of the polypeptides increased only slightly, if at all. Moreover, the ratio of all of the chlorosomal polypeptides remained largely constant. These results show that chlorosomes may incorporate newly synthesized BChlc without concomitant formation of chlorosomal polypeptides. This means that there was no obvious coordination of polypeptide and BChlc synthesis. On this basis, it appears unlikely that one of the chlorosomal polypeptides functions as an apoprotein of a presumed BChlc holochrome complex.     

Chlorosome development in Chloroflexus aurantiacus

The development of chlorosomes was studied in the green phototrophic bacterium Chloroflexus aurantiacus during the adaptation from chemotrophic (aerobiosis in the dark) to phototrophic (anaerobiosis in the light) conditions. Electron micrographs confirmed that chlorosomes were essentially absent from chemotrophic cells. After 5 h of adaptation, however, about 70% of the cells exhibited the presence of chlorosomes and after 19 h essentially all the cells contained chlorosomes. During the first 5 h of adaptation, the number of chlorosomes per µm² of membrane area increased from zero to 37 ± 7, and during the following 40 h to 55 ± 17. The latter phase was characterized by an increase in the chlorosome volume from 36 400 to 91 800 nm³. Chemotrophic cells contained all of the three polypeptides assumed to be localized in the chlorosome envelope. As estimated on the basis of bacteriochlorophyll (BChl) c of chlorosomes, the relative contents of all of the three polypeptides decreased during the adaptation to phototrophic conditions by a factor of about eight. It is proposed that largely empty chlorosome bags are already present in chemotrophic cells and that these as well as subsequently formed chlorosomes are filled up with BChl c. The results are discussed in light of the role of the 5.7 kDa polypeptide in the arrangement of BChl c aggregates within the chlorosome.