Metabolic shift analysis at high cell densities

Abstract: In high cell density cultures it is virtually inevitable that the environment to which the cells are exposed is heterogeneous. Thus, with suspended cultures, individual cells are subject to temporal changes in their environment whereas with aggregated or immobilized cells, the culture can be considered as being formed by a number of subpopulations, each with its own environmental characteristics. In addition, in a high cell density environment, high concentrations of end products may negatively influence the growth rate. This may result in the selection of organisms with an altered metabolic behaviour or with a decreased sensitivity to the adverse effects of the product. We discuss the consequences of this heterogeneity with regard to carbon source metabolism in view of the ability of many bacterial species to adapt to environmental conditions. Selection of variant organisms was found to occur with Clostridium butyricum when grown for a prolonged time in a medium containing approx. I-50 mM glucose. In contrast to the original strain, these variants could sustain a high maximal growth rate in the presence of butyric acid. In addition, they had acquired the capacity to spontaneously form aggregates and were able to carry out a completely solventogenic fermentation. Heterogeneous metabolic activity in aggregated cells is demonstrated with cultures of Lactobacillus laevolacticus , an aggregateforming lactic acid bacterium that converts glucose completely to o-lactate. By using microelectrodes, we show that the fraction of metabolically active cells decreases with increasing aggregate size: in larger aggregates steep pH gradients occur with the effect that only the outer layer of the aggregate is metabolically active, i.e. contributes to lactic acid formation, whereas with smaller aggregates all cells remain active. As a result, the net specific lactic acid production rate of the population as a whole is not invariably increased with increased aggregate size.     

Selective disruption of energy flow from phycobilisomes to Photosystem I

Efficient production of ATP and NADPH by the light reactions of oxygen-evolving photosynthesis demands continuous adjustment of transfer of absorbed light energy from antenna complexes to Photosystem I (PS I) and II (PS II) reaction center complexes in response to changes in light quality. Treatment of intact cyanobacterial cells with N-ethylmaleimide appears to disrupt energy transfer from phycobilisomes to Photosystem I (PS I). Energy transfer from phycobilisomes to Photosystem II (PS II) is unperturbed. Spectroscopic analysis indicates that the individual complexes (phycobilisomes, PS II, PS I) remain functionally intact under these conditions. The results are consistent with the presence of connections between phycobiliproteins and both PS II and PS I, but they do not support the existence of direct contacts between the two photosystems.Abbreviations Chl chlorophyll - EPR electron paramagnetic resonance - NEM N-ethylmaleimide - PBS phycobilisome - PS photosystem     

Experimental and theoretical definition of geminivirus origin of replication

Geminiviruses are plant pathogens that replicate by a rolling-circle mechanism, analogous to that used by several prokaryotic ssDNA replicons. Recent reports provide important progress in understanding the structure and functioning of replication origin from these viruses. We have used these data to propose models for the initiation of replication in dicot- and monocot-infecting geminiviruses.     

The types of bioreactors used for shoots and embryos

Plant regenerated organs such as shoots, bulbs, microtubers, corms, embryos, etc. have been successfully proliferated in the bioreactor. The use of a bioreactor leads to the development of technology suitable for large scale plant propagation. The basic construction and characteristics of various types of bioreactor systems are reviewed in relation to shoot and embryo cultures. A pilot scale 500 liter bioreactor system was applied to the production of large scale Stevia rebaudiana shoots.Abbreviations DW dry weight - EC electrical conductivity - FW fresh weight - ORP oxidation-reduction potential     

The new class 11 transposon Tn163 is plasmid-borne in two unrelated Rhizobium leguminosarum biovar viciae strains

Tn163 is a transposable element identified in Rhizobium leguminosarum bv. viciae by its high insertion rate into positive selection vectors. The 4.6 kb element was found in only one further R. leguminosarum bv. viciae strain out of 70 strains investigated. Both unrelated R. leguminosarum bv. viciae strains contained one copy of the transposable element, which was localized in plasmids native to these strains. DNA sequence analysis revealed three large open reading frames (ORFs) and 38 bp terminal inverted repeats. ORF1 encodes a putative protein of 990 amino acids displaying strong homologies to transposases of class 11 transposons. ORF2, transcribed in the opposite direction, codes for a protein of 213 amino acids which is highly homologous to DNA invertases and resolvases of class II transposons. Homology of ORF1 and ORF2 and the genetic structure of the element indicate that Tn163 can be classified as a class II transposon. It is the first example of a native transposon in the genus Rhizobium. ORF3, which was found not to be involved in the transposition process, encodes a putative protein (256 amino acids) of unknown function. During transposition Tn163 produced direct repeats of 5 bp, which is typical for transposons of the Tn3 family. However, one out of the ten insertion sites sequenced showed a 6 by duplication of the target DNA; all duplicated sequences were A/T rich. Insertion of Tn163 into the sacB gene revealed two hot spots. Chromosomes of different R. leguminosarum bv. viciae strains were found to be highly refractory to the insertion of Tn163.     

Fine-scale temporal variation in recruitment of a temperate demersal fish: the importance of settlement versus post-settlement loss

In order to understand variability in recruitment to populations of benthic and demersal marine species, it is critical to distinguish between the contributions due to variations in larval settlement versus those caused by post-settlement mortality. In this study, fine-scale (1–2 days) temporal changes in recruit abundance were followed through an entire settlement season in a temperate demersal fish in order to determine 1) how dynamic the process of recruitment is on a daily scale, 2) whether settlement and post-settlement mortality are influenced by habitat structure and conspecific density, and 3) how the relationship between settlement and recruitment changes over time. Settlement is considered to be the arrival of new individuals from the pelagic habitat, and recruitment is defined as the number of individuals surviving arbitrary periods of time after settlement. Replicate standardized habitat units were placed in 2 spatial configurations (clumped and randomly dispersed) and monitored visually for cunner (Tautogolabrus adspersus) settlement and recruitment every 1–2 days throughout the settlement season. The process of recruitment in T. adspersus was highly variable at a fine temporal scale. Changes in the numbers of recruits present on habitat units were due to both settlement of new individuals and mortality of animals previously recruited. The relative importance of these two processes appeared to change from day to day. The magnitude of the change in recruit number did not differ between the clumped and random habitats. However, post-settlement loss was significantly greater on randomly dispersed than clumped habitats. During several sampling dates, the extent of the change in recruit abundance was correlated with the density of resident conspecifics; however, on other dates no such relationship appeared to exist. Despite the presence of significant relationships between the change in recruit number and density, there was no evidence of either density-dependent mortality or settlement. Initially, there was a strong relationship between settlement and recruitment; however, this relationship weakened over time. Within 2 months after the cessation of settlement, post-settlement loss was greater than 99%, and no correlation remained between recruitment and the initial pattern of settlement. The results of this study demonstrate that the spatial arrangement of the habitat affects the rate and intensity of post-settlement loss. Counter to much current thinking, this study suggests that in order to understand the population ecology of reef fishes, knowledge of what habitats new recruits use and how mortality varies with structural aspects of the habitats is essential.     

Effects of predatory risk and resource renewal on the timing of foraging activity in a gerbil community

The foraging decisions of animals are often influenced by risk of predation and by the renewal of resources. For example, seed-eating gerbils on sand dunes in the Negev Desert of Israel prefer to forage in the bush microhabitat and during darker hours due to risk of predation. Also, daily renewal of seed resource patches and timing of nightly foraging activity in a depleting environment play important roles in species coexistence. We examined how these factors influence the timing of gerbil foraging by quantifying foraging activity in seed resource patches that we experimentally renewed hourly during the night. As in previous work, gerbils showed strong preference for the safe bush microhabitat and foraged less in response to high levels of illumination from natural moon light and from artificial sources. We demonstrate here for the first time that gerbils also responded to temporal and spatial heterogeneity in predatory risk through their timing of activity over the course of each night. Typically, gerbils concentrated their activity early in the night, but this changed with moon phase and in response to added illumination. These results can be understood in terms of the nature of patch exploitation by gerbils and the role played by the marginal value of energy in determining the cost of predation. They further show the dynamic nature of gerbil foraging decisions, with animals altering foraging efforts in response to time, microhabital, moon phase, illumination, and resource availability.     

Involvement of putrescine in the inductive rooting phase of poplar shoots raised in vitro

Micropropagated poplar shoots rooted 100% on a rooting medium (A) containing NAA, but they did not root in the absence of auxin (NA). Putrescine, but not spermidine and spermine, promoted rooting up to 42% when added to the NA medium. Cyclohexylamine (CHA), an inhibitor of spermine synthase, also promoted (up to 36%) rooting in the absence of auxin. The inhibitors of polyamine biosynthesis DFMA (α-difluoromethylarginine) and DFMO (α-difluoromethylomithine), aminoguanidine (AG) and methylglyoxal-bis-guanylhydrazone (MGBG), inhibited rooting when applied in the presence of auxin and had no effect in its absence.
The rooting inductive phase (in the presence of auxin) was determined by periodical transfer of shoots from A to NA medium, and by changes in peroxidase activity, to be 7 h. Putrescine (not spermidine and spermine) accumulated to a maximum during the inductive phase. Both putrescine and CHA promoted rooting on NA medium when applied during the first 7 h. In contrast DFMA and AG inhibited rooting during this period. The results point to the involvement of putrescine and its Δ¹-pyrroline pathway, in the inductive phase of rooting in poplar shoots.