Overexpression of cloned genes using recombinant Escherichia coli regulated by a T7 promoter: II. Two-stage continuous cultures and model simulations

A two-stage culture strategy was studied for continuous high-level production of a foreign protein in the chemically inducible T7 expression system. The first stage is dedicated to the maintenance of plasmid-bearing cells and the second stage to the target protein synthesis by induction of cells coming from the first stage. On entering the second stage, recombinant cells undergo a gradual induction of the target gene expression. These plasmid-bearing cells experience dynamic changes in intracellular compositions and specific growth rates with their individual residence times. Therefore, the overall cultural characteristics in the production stage are really averages of the contributions from the various cells with different residence times. The behavior of the two-stage culture is described by a model, which accounts for dynamic variations of cell growth and protein synthesis rates with cell residence times. Model simulations were compared with experimental results at a variety of operating conditions such as inducer concentration and dilution rate. This model is useful for understanding the behavior of two-stage continuous cultures. (c) 1993 John Wiley & Sons, Inc.     

New preparation techniques for molecular and in‐situ analysis of ancient organic micro‐ and nanostructures

Organic microfossils preserved in three dimensions in transparent mineral matrices such as cherts/quartzites, phosphates, or carbonates are best studied in petrographic thin sections. Moreover, microscale mass spectrometry techniques commonly require flat, polished surfaces to minimize analytical bias. However, contamination by epoxy resin in traditional petrographic sections is problematic for the geochemical study of the kerogen in these microfossils and more generally for the in situ analysis of fossil organic matter. Here, we show that epoxy contamination has a molecular signature that is difficult to distinguish from kerogen with time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS). This contamination appears pervasive in organic microstructures embedded in micro‐ to nano‐crystalline carbonate. To solve this problem, a new semi‐thin section preparation protocol without resin medium was developed for micro‐ to nanoscale in situ investigation of insoluble organic matter. We show that these sections are suited for microscopic observation of Proterozoic microfossils in cherts. ToF‐SIMS reveals that these sections are free of pollution after final removal of a <10 nm layer of contamination using low‐dose ion sputtering. ToF‐SIMS maps of fragments from aliphatic and aromatic molecules and organic sulfur are correlated with the spatial distribution of organic microlaminae in a Jurassic stromatolite. Hydrocarbon‐derived ions also appeared correlated with kerogenous microstructures in Archean cherts. These developments in analytical procedures should help future investigations of organic matter and in particular, microfossils, by allowing the spatial correlation of microscopy, spectroscopy, precise isotopic microanalyses, and novel molecular microanalyses such as ToF‐SIMS.     

Empirical relationships to estimate asymptotic length, length at first maturity and length at maximum yield per recruit in fishes, with a simple method to evaluate length frequency data

Empirical relationships are presented to estimate in fishes, asymptotic length (L∞) from maximum observed length (L_max), length at first maturity (L_m) from L_∞, life span (t_max) from age at first maturity (t_m), and length at maximum possible yield per recruit (L_opt) from L_∞ and from L_m, respectively. The age at L_opt is found to be a good indicator of generation time in fishes. A spreadsheet containing the various equations can be downloaded from the Internet at http://www.fishbase.org/download as popdynJFB.zip. A simple method is presented for evaluation of length–frequency data in their relationship to L_∞, L_m and L_opt. This can be used to evaluate the quality of the length–frequency sample and the status of the population. Three examples demonstrate the usefulness of this method. 2000 The Fisheries Society of the British Isles     

Citric acid production and morphology of Aspergillus niger as functions of the mixing intensity in a stirred tank and a tubular loop bioreactor

The relationship between Aspergillus niger morphology and citric acid production was investigated in two reactor systems with different configurations, a tubular loop and a stirred tank bioreactor, with operating volumes of 6 and 8 dm³, respectively. Morphology was quantified by image analysis. In each system, morphology, characterized by the parameter P (mean convex perimeter of clumps), and citric acid production, were agitation-dependent and closely linked. Increased agitation caused a reduction of clump sizes and results when both reactors demonstrate that the parameter P should not exceed a threshold value in order to achieve increased productivities. The results obtained from the two reactors were in agreement, both qualitatively and quantitatively. Reducing the fundamentally different mixing conditions of the two bioreactors to the order of the dimensionless mixing parameter relative mixing time (τ_m), results showed that the loop simulated the stirred tank. Also, relationships valid for one system accurately described the results obtained from the other system, demonstrating the validity of the relationship between morphology and productivity for the particular fermentation, regardless of the reactor type. Previous attempts to evaluate the use of loop configurations as scale-up tools and their performance as bioreactors, neglected the morphology of the producer micro-organisms. This study demonstrated the close link between morphology and productivity for citric acid production by A. niger, and identified a morphology parameter that was used successfully to characterize the process performance.     

Psb30 contributes to structurally stabilise the Photosystem II complex in the thermophilic cyanobacterium Thermosynechococcus elongatus

A deletion mutant that lacks the Psb30 protein, one of the small subunits of Photosystem II, was constructed in a Thermosynechococcus elongatus strain in which the D1 protein is expressed from the psbA₃ gene (WT*). The ΔPsb30 mutant appears more susceptible to photodamage, has a cytochrome b₅₅₉ that is converted into the low potential form, and probably also lacks the PsbY subunit. In the presence of an inhibitor of protein synthesis, the ?Psb30 lost more rapidly the water oxidation function than the WT* under the high light conditions. These results suggest that Psb30 contributes to structurally and functionally stabilise the Photosystem II complex in preventing the conversion of cytochrome b₅₅₉ into the low potential form. Structural reasons for such effects are discussed.     

Identification and quantification of viable Bifidobacterium breve strain Yakult in human faeces by using strain-specific primers and propidium monoazide

Aims: To develop a quick and accurate PCR‐based method to evaluate viable Bifidobacterium breve strain Yakult (BbrY) in human faeces. Methods and Results: The number of BbrY in faeces was detected by using strain‐specific quantitative real‐time PCR (qPCR) derived from a randomly amplified polymorphic DNA analysis. And using propidium monoazide (PMA) treatment, which combined a DNA‐intercalating dye for covalently linking DNA in dead cells and photoactivation, only viable BbrY in the faeces highly and significantly correlated with the number of viable BbrY added to faecal samples within the range of 10⁵–10⁹ cells per g of faeces was enumerated. After 11 healthy subjects ingested 10·7 log CFU of BbrY daily for 10 days, 6·9 (±1·5) log CFU g^?1 [mean (±SD)] of BbrY was detected in faeces by using strain‐specific transgalactosylated oligosaccharide–carbenicillin (T‐CBPC) selective agar medium. Viable BbrY detected by qPCR with PMA treatment was 7·5 (±1·0) log cells per g and the total number (viable and dead) of BbrY detected by qPCR without PMA treatment was 8·1 (±0·8) log cells per g. Conclusions: Strain‐specific qPCR with PMA treatment evaluated viable BbrY in faeces quickly and accurately. Significance and Impact of the Study: Combination of strain‐specific qPCR and PMA treatment is useful for evaluating viable probiotics and its availability in humans.     

Local Rather than Global Folding Enables the Lead-dependent Activity of the 8-17 Deoxyribozyme: Evidence from Contact Photo-crosslinking

The 8-17 deoxyribozyme is an in vitro selected enzyme capable of sequence-specific cleavage of RNA. While selected to be a magnesium and zinc-utilizing enzyme, the 8-17 DNAzyme has been shown to utilize lead for its catalysis. Fluorescence-based experiments have indicated that the magnesium- and zinc-utilizing versions of the DNAzyme-substrate complex need to form a defined tertiary structure to be active, but no such global folding is required for the lead-mediated activity. Here, we have investigated this phenomenon, including the use of contact photo-crosslinking to map the tertiary fold of the lead-dependent DNAzyme. While our results recapitulate that global folding is not required for the lead activity, they reveal strikingly distinct lead-mediated modes of activity under conditions of low versus moderate solution ionic strength. Even in very low salt buffers, where no global folding of the 8-17 DNAzyme occurs, the active site of the enzyme appears to form a distinct local fold, one that cannot be modelled easily by DNA/RNA constructs that preserve key sequence and secondary structure features of the active site.     

Plant nitrogen dynamics and nitrogen-use strategies under altered nitrogen seasonality and competition

BACKGROUND AND AIMS: Numerous studies have examined the effects of climatic factors on the distribution of C(3) and C(4) grasses in various regions throughout the world, but the role of seasonal fluctuations in temperature, precipitation and soil N availability in regulating growth and competition of these two functional types is still not well understood. This report is about the effects of seasonality of soil N availability and competition on plant N dynamics and N-use strategies of one C(3) (Leymus chinensis) and one C(4) (Chloris virgata) grass species. METHODS: Leymus chinensis and C. virgata, two grass species native to the temperate steppe in northern China, were planted in a monoculture and a mixture under three different N seasonal availabilities: an average model (AM) with N evenly distributed over the growing season; a one-peak model (OM) with more N in summer than in spring and autumn; and a two-peak model (TM) with more N in spring and autumn than in summer. KEY RESULTS: The results showed that the altered N seasonality changed plant N concentration, with the highest value of L. chinensis under the OM treatment and C. virgata under the TM treatment, respectively. N seasonality also affected plant N content, N productivity and N-resorption efficiency and proficiency in both the C(3) and C(4) species. Interspecific competition influenced N-use and resorption efficiency in both the C(3) and C(4) species, with higher N-use and resorption efficiency in the mixture than in monoculture. The C(4) grass had higher N-use efficiency than the C(3) grass due to its higher N productivity, irrespective of the N treatment or competition. CONCLUSIONS: The observations suggest that N-use strategies in the C(3) and C(4) species used in the study were closely related to seasonal dynamics of N supply and competition. N seasonality might be involved in the growth and temporal niche separation between C(3) and C(4) species observed in the natural ecosystems.