Selection of anthocyanin-accumulating potato (Solanum tuberosum L.) cell lines from calli derived from seedlings produced by gamma-irradiated seeds

Callus cell lines of potato (Solanum tuberosum L. cv. Zarevo) were obtained from seedlings germinated from gamma-irradiated seeds (200 Gy). Some of these cell lines produce red-violet pigments which were identified as acylated anthocyanins. The major anthocyanin was determined to be peonidin 3-O-[6-O-(4-O-E-p-coumaroyl-rhamnosyl)-glucoside]-5-O-glucoside (peonanin). Single cell-derived protoclones from non-pigmented protoplasts sometimes also gave rise to pigmented cell clusters thus indicating that the changes in the expression of the anthocyanin pathway can also occur after the stage of initial callus induction.     

Cellulosomics, a gene-centric approach to investigating the intraspecific diversity and adaptation of Ruminococcus flavefaciens within the rumen

Background

The bovine rumen maintains a diverse microbial community that serves to break down indigestible plant substrates. However, those bacteria specifically adapted to degrade cellulose, the major structural component of plant biomass, represent a fraction of the rumen microbiome. Previously, we proposed scaC as a candidate for phylotyping Ruminococcus flavefaciens, one of three major cellulolytic bacterial species isolated from the rumen. In the present report we examine the dynamics and diversity of scaC-types both within and between cattle temporally, following a dietary switch from corn-silage to grass-legume hay. These results were placed in the context of the overall bacterial population dynamics measured using the 16S rRNA.

Principal Findings

As many as 117 scaC-types were estimated, although just nineteen were detected in each of three rumens tested, and these collectively accounted for the majority of all types present. Variation in scaC populations was observed between cattle, between planktonic and fiber-associated fractions and temporally over the six-week survey, and appeared related to scaC phylogeny. However, by the sixth week no significant separation of scaC populations was seen between animals, suggesting enrichment of a constrained set of scaC-types. Comparing the amino-acid translation of each scaC-type revealed sequence variation within part of the predicted dockerin module but strong conservation in the N-terminus, where the cohesin module is located.

Conclusions

The R. flavefaciens species comprises a multiplicity of scaC-types in-vivo. Enrichment of particular scaC-types temporally, following a dietary switch, and between fractions along with the phylogenetic congruence suggests that functional differences exist between types. Observed differences in dockerin modules suggest at least part of the functional heterogeneity may be conferred by scaC. The polymorphic nature of scaC enables the relative distribution of R. flavefaciens strains to be examined and represents a gene-centric approach to investigating the intraspecific adaptation of an important specialist population.     

Specialized cell surface structures in cellulolytic bacteria.

The cell surface topology of various gram-negative and -positive, anaerobic and aerobic, mesophilic and thermophilic, cellulolytic and noncellulolytic bacteria was investigated by scanning electron microscopic visualization using cationized ferritin. Characteristic protuberant structures were observed on cells of all cellulolytic strains. These structures appeared to be directly related to the previously described exocellular cellulase-containing polycellulosomes of Clostridium thermocellum YS (E. A. Bayer and R. Lamed, J. Bacteriol. 167:828-836, 1986). Immunochemical evidence and lectin-binding studies suggested a further correlation on the molecular level among cellulolytic bacteria. The results indicate that such cell surface cellulase-containing structures may be of general consequence to the bacterial interaction with and degradation of cellulose.     

Host Cell Growth in the Presence of the Thermosensitive Drug Resistance Factor, Rts1

We have confirmed and extended the observation of Terawaki et al. that the R factor, Rts1, alters the growth of its host at 42 C. In all media tested there was a period during which total cell numbers increased linearly, while viable counts remained constant. During this period the rate of precursor incorporation per cell particle into deoxyribonucleic acid, ribonucleic acid, and protein declined steadily. These patterns were a consequence of the accumulation of increasing numbers of cells which had lost colony-forming ability. A temperature shiftdown experiment showed that the colony formers could, after a lag, go on to divide normally, whereas most of the noncolony formers could not undergo even a limited number of divisions after shiftdown. The number of normal divisions which occurred after shiftup of Rts1 cells to 42 C was medium dependent. In rich medium there were, on the average, two or three doublings; in glucose medium, one; and in glycerol medium, only a fraction of a doubling. Even in glucose medium, however, no increase in viable counts was observed during growth at 42 C if the cells were first starved for glucose for 1 h at 42 C. A temperature shiftdown from 42 C to 27 C during glucose starvation reversed the effect of starvation at 42 C alone. These results are consistent with the hypothesis that the thermosensitive Rts1 component(s) responsible for the host effects is present at permissive temperature, but can undergo a reversible temperature-induced alteration which then interferes with some essential host function. The detrimental effects of this R factor on its host were also reflected in a heightened sensitivity to kanamycin and actinomycin D at 42 C. Electron microscope observations revealed changes in the appearance of the cell membrane. Membranous invaginations were noted at discrete sites in the cell.