Late Lag Research Articles

Cyanobacteria in late lag and exponential phases suit different pre-chlorination and coagulation strategies.

Pre-chlorination is usually applied to facilitate coagulation process when treating cyanobacteria-laden source waters. However, the responses of cyanobacteria after chlorination were varied in different growth phases, hence how these differences affect the coagulation is not clear. In this study, Microcystis aeruginosa (M. aeruginosa) in late lag and exponential phases were selected for comparing the remove effect of cyanobacterial coagulation after varied doses (0, 0.5, 1.0 and 2.0 mg/L) of pre-chlorination. Results showed that the M. aeruginosa in late lag phase held higher removal efficiencies (48-86%) than those in exponential phase (1-60%). Lower pH and buoyancy, higher Zeta potential and hydrophobicity were beneficial to its coagulation. The application of 0.5-2.0 mg/L pre-chlorination in late lag phase did not promote its coagulation, mainly due to the impediment by released hydrophilic humus. In contrast, the remove efficiency increased about 40% with the addition of 2.0 mg/L chlorine and 15 mg/L polyaluminum chloride (PACl) in exponential phase. The increased macromolecular organic matters, Zeta potential and decreased buoyancy made for this promotion. In addition, the removal of extracellular organic matters, aromatics and microcystin was not that productive after coagulation in exponential phase, but both of chlorination and coagulation could reduce microcystin in late lag phase. The findings suggest that optimizing the application methods of pre-chlorine and coagulant according to the growth phases is necessary for cyanobacterial control.

Nutrient Consumption Patterns of Saccharomyces cerevisiae and Their Application in Fruit Wine Fermentation

This study aimed to evaluate the nutritional requirements of Saccharomyces cerevisiae to improve low ethanol production in some fruit wines. The growth kinetics, ethanol production and nutrient requirements of S. cerevisiae were analyzed in chemically defined media. The results revealed that Ca2⁺, Fe2⁺, Co2⁺, Mo2⁺, Cu2⁺ and BO₃3− were predominantly utilized during the late lag phase, whereas free amino acids, nicotinic acid, calcium pantothenate, Na⁺ and Mg2⁺ were mainly consumed during the logarithmic phase. Compared with the control medium, supplementation with threonine, inositol, calcium pantothenate, thiamine hydrochloride, riboflavin, biotin, MgSO4 or KH2PO4 significantly increased the ethanol content by 1.10-fold (p < 0.05). Furthermore, adding key nutrients to noni-, guava- and mango juice significantly shortened the fermentation time and increased the final alcohol content of the fruit wines (p < 0.05). This study provides scientific insights and effective methods for shortening fermentation time and increasing alcohol content with S. cerevisiae in some fruit wines.

Biological characteristics of marine Streptomyces SK3 and optimization of cultivation conditions for production of compounds against Vibiriosis pathogen isolated from cultured white shrimp (Litopenaeus vannamei).

Antibiotic resistance in shrimp farms has emerged as an extremely serious situation worldwide. The main aim of this study was to optimize the cultural conditions for producing new antibiotic agents from marine Streptomyces species. Streptomyces SK3 was isolated from marine sediment and was identified by its 16S rDNA as well as biochemical characteristics. This microbe produced the highest concentration of bioactive secondary metabolites (BSMs) when cultured in YM medium (YM/2). It produced the maximum total protein (41.8 ± 6.36 mg/ml) during the late lag phase period. The optimum incubation temperature was recorded at 30 °C; BSMs were not produced at ≤10 °C within an incubation period of 3-4 days. The suitable agitation speed was found to be 200 rpm with pH 7.00. The proper carbon, nitrogen, and trace elements supplementation consisted of starch, malt extract, calcium carbonate (CaCO3), and magnesium sulfate (MgSO4). The ethyl acetate extract was found to act strongly against three vibriosis pathogens, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio vunificus, as indicated by the inhibition zones at 34.5, 35.4, and 34.3 mm, respectively. The extract showed the strongest anti-V. harveyi activity, as indicated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 0.101 ± 0.02 and 0.610 ± 0.04 mg/ml, respectively. Basic chemical investigation of the crude extract using thin layer chromatography (TLC), bioautography, liquid chromatography tandem mass spectrometry (LC‒MS/MS), Fourier transform infrared spectroscopy (FTIR), and proton nuclear magnetic resonance (1H-NMR) revealed that the active components were the terpenoid and steroid groups of compounds. They showed carboxylic acid and ester functions in their molecules.

Chlorination for Microcystis aeruginosa in the Late Lag Phase: Higher Inactivation Efficiency and Lower Environmental Risk

Chlorine is often employed to dispose cyanobacteria-laden waters for inactivating cells or improving its removal efficiency. However, previous studies mostly focused on cyanobacterial blooms occurring in the exponential or stationary phase, and thus, cyanobacterial characteristics in the unblooming stage (lag phase) and their influence on chlorination were not well understood. Herein, Microcystis aeruginosa in late lag and exponential phases with the same cell density were selected to simulate unblooming and blooming stages, respectively, and their response to chlorination was investigated. Compared with the exponential phase, results showed that cyanobacteria in the late lag phase consumed lower chlorine but produced higher intracellular reactive oxygen species (ROS). Then, higher rate constants of photosynthetic activity inactivation (kf) and membrane damage (km) were found in the late lag phase. Chlorine of >1.0 mg L–1 could degrade microcystin in this phase, and microcystin could be oxidized to below 1.0 μg L–1 by 2.0 mg L–1 chlorine. Besides, lower extracellular dissolved organic carbon (DOC), UV absorbance at 254 nm (UV254), and their variation range were also observed in the late lag phase. These findings indicated that the chlorination performance in the two phases is different, and thus, it is necessary to reduce the applied dosage of chlorine to treat cyanobacteria-laden source waters in the (late) lag phase.

Higher sensitivity to Cu2+ exposure of Microcystis aeruginosa in late lag phase is beneficial to its control

Cyanobacterial blooms are always treated in exponential phase, which demands high dosages of algicides (e.g., CuSO4). Actually, cyanobacterial blooms in late lag phase exhibit low cell-density and specific physiological/biochemical characteristics, implying the possibility of controlling blooms in a more efficient and economical way with CuSO4 treatment if cyanobacterial cells in late lag phase can be treated. In this study, the outbreakof a Microcystis bloom was simulated, and Microcystis samples in late lag and exponential phases were treated with CuSO4. The results showed that M. aeruginosa in late lag phase had a higher ratio of dividing-cells, Fv/Fm and intracellular total organic carbon content (TOC) than that in exponential phase, indicating that its metabolic activity was vigorous. M. aeruginosa in late lag phase could more easily be blocked, since a higher decrease in chlorophyll-a, Fv/Fm and membrane integrity occurred under the same dosages of CuSO4 exposure compared to M. aeruginosa in exponential phase. Meanwhile, microcystin release in late lag phase was less than that in exponential phase. Moreover, higher sensitivity in late lag phase was confirmed at the individual level, as the photosynthesis related genes psaB and rbcL were more down-regulated than those in exponential phase. In general, cyanobacteria in late lag phase exhibited higher sensitivity to CuSO4, indicating that CuSO4 treatments in late lag phase can achieve a higher control efficiency and fewer release of microcystin with low-dosages algicide. Hence, it is a more environmentally friendly strategy to control cyanobacterial blooms than the traditional strategy applied in exponential phase.

A glimpse of potential transposable element impact on adaptation of the industrial yeast Saccharomyces cerevisiae.

The adapted industrial yeast strain Saccharomyces cerevisiae NRRL Y-50049 is able to in situ detoxify major toxic aldehyde compounds derived from sugar conversion of lignocellulosic biomass while producing ethanol. Pathway-based studies on its mechanisms of tolerance have been reported previously, however, little is known about transposable element (TE) involvement in its adaptation to inhibitory compounds. This work presents a comparative dynamic transcription expression analysis in response to a toxic treatment between Y-50049 and its progenitor, an industrial type strain NRRL Y-12632, using a time-course study. At least 77 TEs from Y-50049 showed significantly increased expression compared with its progenitor, especially during the late lag phase. Sequence analysis revealed significant differences in TE sequences between the two strains. Y-50049 was also found to have a transposons of yeast 2 (Ty2) long terminal repeat-linked YAT1 gene showing significantly higher copy number changes than its progenitor. These results raise awareness of potential TE involvement in the adaptation of industrial yeast to the tolerance of toxic chemicals.

Characterization of a broad spectrum bacteriocin produced by Lactobacillus plantarum MXG-68 from Inner Mongolia traditional fermented koumiss.

An agar well diffusion assay (AWDA) was used to isolate a high bacteriocin-producing strain with a broad spectrum of antibacterial activity, strain MXG-68, from Inner Mongolia traditional fermented koumiss. Lactobacillus plantarum MXG-68 was identified by morphological, biochemical, and physiological characteristics and 16S rDNA analysis. The production of antibacterial substance followed a growth-interrelated model, starting at the late lag phase of 4h and arriving at a maximum value in the middle of the stationary phase at 24h. Antibacterial activity was abolished or decreased in the presence of pepsin, chymotrypsin, trypsin, proteinase, and papain K. The results showed that antibacterial substances produced by L. plantarum MXG-68 were proteinaceous and could thus be classified as the bacteriocin, named plantaricin MXG-68. The molar mass of plantaricin MXG-68 was estimated to be 6.5kDa, and the amino acid sequence of its N-terminal was determined to be VYGPAGIFNT. The mode of plantaricin MXG-68 action was determined to be bactericidal. Bacteriocin in cell-free supernatant (CFS) at pH7 was stable at different temperatures (60°C, 80°C, 100°C, 121°C for 30min; 4°C and - 20°C for 30days), as well as at pH2.0-10.0. Antibacterial activity maintained stable after treatment with organic solvents, surfactants, and detergents but increased in response to EDTA. Response surface methodology (RSM) revealed the optimum conditions of bacteriocin production in L. plantarum MXG-68, and the bacteriocin production in medium optimized by RSM was 26.10% higher than that in the basal MRS medium.

Keratinase-producing fungi from local environmental samples of Far South Thailand and their efficiency in hydrolyzing keratinous wastes

Samples from around BangoRana, BangoAsae, and Tanod, Narathiwat Province, Thailand were screened for keratinase-producing fungi. By using Horikoshi media and keratin (1%) agar plate, 3 isolates of fungi were shown to produce wider clear zone, and they were Aspergillus sp., Penicillin sp. and Cladosporium sp., and designated as Aspergillus KF1, Penicillin KF2 and Cladosporium KF3. Each isolate was growth in Feather Meal Medium with 37°C incubation in shaking condition. Results showed that these fungi had typical growth profile. Extracellular keratinase profile for Penicillium KF2, the keratinase release began at the late lag phase (1 U/ml), whereas those of Aspergillus KF1, and Cladosporium KF3 started at the log phase (U/ml: 3.9 and 2.9). Maximal keratinase production was observed in the fungi culture on day 4 and day 5 upon shaking incubation at 37°C. Different activities were observed in U/ml likes 70.7 for Aspergillus KF1; 83.5, Penicillium KF2; and 72.4, Cladosporium KF3. Aspergillus KF1 and Penicillin FK2 reached their maximum on day 4 (CFU/ml: 8.9 × 106 and 8.4 × 106) Cladosporium FK3 on day 5 were 8.95 × 106 CFU/ml. Keratinases were expected to be of primary metabolites, and hence best harvested at the beginning of stationary phase.

Bistability in myo -Inositol Utilization by Salmonella enterica Serovar Typhimurium

The capability of Salmonella enterica serovar Typhimurium strain 14028 (S. Typhimurium 14028) to utilize myo-inositol (MI) is determined by the genomic island GEI4417/4436 carrying the iol genes that encode enzymes, transporters, and a repressor responsible for the MI catabolic pathway. In contrast to all bacteria investigated thus far, S. Typhimurium 14028 growing on MI as the sole carbon source is characterized by a remarkable long lag phase of 40 to 60 h. We report here that on solid medium with MI as the sole carbon source, this human pathogen exhibits a bistable phenotype characterized by a dissection into large colonies and a slow-growing bacterial background. This heterogeneity is reversible and therefore not caused by mutation, and it is not observed in the absence of the iol gene repressor IolR nor in the presence of at least 0.55% CO(2). Bistability is correlated with the activity of the iolE promoter (P(iolE)), but not of P(iolC) or P(iolD), as shown by promoter-gfp fusions. On the single-cell level, fluorescence microscopy and flow cytometry analysis revealed a gradual switch of P(iolE) from the "off" to the "on" status during the late lag phase and the transition to the log phase. Deletion of iolR or the addition of 0.1% NaHCO(3) induced an early growth start of S. Typhimurium 14028 in minimal medium with MI. The addition of ethoxyzolamide, an inhibitor of carboanhydrases, elongated the lag phase in the presence of bicarbonate. The positive-feedback loop via repressor release and positive induction by bicarbonate-CO(2) might allow S. Typhimurium 14028 to adapt to rapidly changing environments. The phenomenon described here is a novel example of bistability in substrate degradation, and, to our knowledge, is the first demonstration of gene regulation by bicarbonate-CO(2) in Salmonella.

Improving the production of a thermostable amidase through optimising IPTG induction in a highly dense culture of recombinant Escherichia coli

The production of a novel thermostable amidase (EC 3.5.1.4) from Geobacillus pallidus RAPc8 using recombinant Escherichia coli BL21 (DE3) was investigated. Volumetric and specific enzyme activities were investigated in relation to inducer concentration in a batch process using a defined medium with glucose as the carbon source. While IPTG is routinely used to induce expression of genes under the control of lac promoter, the impact of high biomass concentration on IPTG induction has not been reported rigorously. In this study, biomass production was unaffected by IPTG concentration across the range 0–1000 μM. Induction of recombinant protein expression by 400 μM IPTG at late lag phase of growth (3rd hour) inhibited cell growth while induction at early exponential phase of growth (5th hour) gave a 3 fold increase in volumetric amidase activity compared to induction at mid exponential phase (8th hour). Protein production increased by a factor of two with IPTG addition, independent of IPTG concentration in the range of 40–1000 μM. Amidase activity, measured on a volumetric basis and relative to protein and biomass concentrations, increased with increasing IPTG concentration up to 400 μM. While inducer concentrations are typically reported on a volumetric basis, their mode of action is consistent with a biomass dependence. Analysis of the data across a range of biomass concentration confirmed that induction was a function of inducer concentration per unit biomass. The amidase enzyme was predominantly soluble and cytoplasmic with less than 3% retained within the cell debris.

Effects of monoamine neuromediators on the growth-related variables of Escherichia coli K-12

The monoamine neuromediators serotonin (5-HT), histamine, dopamine (DA), and norepinephrine (NE), added to an Escherichia coli K-12 strain MC 4100 culture upon inoculation, stimulate cell proliferation (determined from CFU formation) and biomass accumulation (monitored nephelometrically) during the late lag phase and the early exponential growth phase. These effects are less significant in the late exponential and stationary phase cultures. According to the concentration dependence of the stimulatory effects, the neuromediators can be classified into two groups: (i) the catecholamines DA and NE, whose effects increase almost linearly with increasing concentrations within the range of 0.1-100 microM, and (ii) histamine and 5-HT, which are characterized by bell-shaped concentration dependence curves with maxima at 0.1 (histamine) and 1 microM (5-HT). On an agar-containing medium, the growing E. coli population includes solitary cells and compact cell groups (microcolonies). In this system, both tested catecholamines exert a relatively weak stimulatory influence that manifests itself as an increase in the number of both solitary cells and cell groups, and occurs at concentrations of 10 microM and higher. In analogy to the culture grown on the liquid medium, 5-HT and histamine are distinguished by nonlinear concentration dependence curves: their effects peak at 0.1 microM (histamine) or 1 microM (5-HT); an increase in the neuromediator concentrations results in a decrease in effects that are enhanced by further increasing the concentrations to the submillimolar range. DA increases the percentage of solitary cells, whereas the other tested amines promote cell group formation. The results are interpreted in terms of specific (probably receptor-dependent) mechanisms of action of the neuromediators involved.

Effect of tobacco deprivation on the attentional blink in rapid serial visual presentation

When two targets are imbedded in rapid serial visual presentation (RSVP), identification of the second target (T2) is impaired if it occurs within 500 ms of the first target (T1). This attentional blink (AB) is thought to involve interference of resources in processing T1 and T2. The deleterious effect of tobacco deprivation on attention has been documented, but no studies have examined the AB. Nonsmokers (n=30), 12-h tobacco-deprived smokers (n=30), and nondeprived smokers (n=30) were randomly assigned to perform the RSVP with one of three stimulus-duration conditions (96, 113, or 130 ms). Participants completed 48 RSVP trials. Each trial consisted of 16 individually presented words (T1, T2, and 14 distractors), and T2 lagged T1 at serial positions 1-8. Participants verbalized T1 and T2 in order immediately after each trial. Identification of T2 (for correct T1 trials) was impaired at early versus late lag positions, which was especially pronounced in the most difficult (96 ms) condition. There was no evidence for group differences on the AB; however, deprived smokers were worse identifying T1 in the 113-ms condition. These results suggest that the AB is influenced by stimulus duration, but not by 12 h of tobacco deprivation.

Effect of Growth Phase on Survival of Bromegrass Suspension Cells Following Cryopreservation and Abiotic Stresses

Cryopreservation is a practical method of preserving plant cell cultures and their genetic integrity. It has long been believed that cryopreservation of plant cell cultures is best performed with cells at the late lag or early exponential growth phase. At these stages the cells are small and non-vacuolated. This belief was based on studies using conventional slow prefreezing protocols and survival determined with fluorescein diacetate staining or 2,3,5-triphenyltetrazolium chloride assays. This classical issue was revisited here to determine the optimum growth phase for cryopreserving a bromegrass (Bromus inermis) suspension culture using more recently developed protocols and regrowth assays for determination of survival. Cells at different growth phases were cryopreserved using three protocols: slow prefreezing, rapid prefreezing and vitrification. Stage-dependent trends in cell osmolarity, water content and tolerance to freezing, heat and salt stresses were also determined. In all cases survival was assayed by regrowth of cells following the treatments. Slow prefreezing and rapid prefreezing protocols resulted in higher cell survival compared with the vitrification method. For all the protocols used, the best regrowth was obtained using cells in the late exponential or early stationary phase, whereas lowest survival was obtained for cells in the late lag or early exponential phase. Cells at the late exponential phase were characterized by high water content and high osmolarity and were most tolerant to freezing, heat and salt stresses, whereas cells at the early exponential phase, characterized by low water content and low osmolarity, were least tolerant. The results are contrary to the classical concept which utilizes cells in the late lag or early exponential growth phase for cryopreservation. The optimal growth phase for cryopreservation may depend upon the species or cell culture being cryopreserved and requires re-investigation for each cell culture. Stage-dependent survival following cryopreservation was proportionally correlated with the levels of abiotic stress tolerance in bromegrass cells.

The lag phase and G1 phase of a single yeast cell monitored by Raman microspectroscopy

We optically trapped a single yeast cell for up to 3 h and monitored the changes in the Raman spectra during the lag phase of its growth and the G1 phase of its cell cycle. A non-budding cell (corresponding either to the G0 or G1 phase) was chosen for each experiment. During the lag phase, the cell synthesises new proteins and lipids and the observed behaviour of the peaks corresponding to these constituents as well as those of RNA served as a sensitive indicator of the adaptation of the cell to its changed environment. Temporal behaviour of the Raman peaks observed was different in the lag phase as compared to the late lag phase. Two different laser wavelengths were applied to study the effect of long-term optical trapping on the living cells. Yeast cells killed either by boiling or by a chemical protocol were also trapped for a long time in a single beam optical trap to understand the effect of optical trapping on the behaviour of observed Raman peaks. The changes observed in the Raman spectra of a trapped yeast cell in the late G1 phase or the beginning of S phase corresponded to the growth of a bud. Copyright © 2006 John Wiley & Sons, Ltd.

The influence of the growth phase of enteric bacteria on electrotransformation with plasmid DNA.

Salmonella typhimurium LB5000 and Escherichia coli JM109 were transformed by electroporation. In accordance with the chemical transformation methods, the growth phase of these electrocompetent bacteria had a strong impact on transformation efficiency. Survival of bacteria after the high-voltage electrical pulse was also influenced by the growth phase. Both bacterial species were most successfully electrotransformed when microbial cells were harvested at the late lag phase. The second optimum for transformation reached E. coli cells in the mid-exponential and S. typhimurium cells in the late exponential phase. Transformation efficiencies ranged from 3.4 x 10(4) to 2.7 x 10(5) transformants per microgram DNA in the case of S. typhimurium and from 2.8 x 10(2) to 8.8 x 10(5) transformants per microgram DNA in the case of E. coli. Survival of cells after the electrical pulse in late lag and late exponential phases was about 20% higher than during other phases of growth. Preparing electrocompetent cells from later phases of their growth is more useful for practice, because it provides more biomass with good yield of transformants.

Exopolysaccharide production in biofilms: substratum activation of alginate gene expression by Pseudomonas aeruginosa.

Reporter gene technology was employed to detect the activity of an alginate promoter of Pseudomonas aeruginosa when the organism was grown as a biofilm on a Teflon mesh substratum and as planktonic cells in liquid medium. Alginate biosynthetic activity was determined with a mucoid cell line derived from a cystic fibrosis isolate and containing an alginate algC promoter fused to a lacZ reporter gene. Reporter activity was demonstrated with chromogenic and fluorogenic substrates for beta-galactosidase. Expression of algC was shown to be upregulated in biofilm cells compared with planktonic cells in liquid medium. Gene up-expression correlated with alginate biosynthesis as measured by Fourier transform infrared spectroscopy, uronic acid accumulation, and alginate-specific enzyme-linked immunosorbent assay. The algC promoter was shown to have maximum activity in planktonic cultures during the late lag and early log phases of the cell growth cycle. During a time course experiment, biofilm algC activity exceeded planktonic activity except during the period immediately following inoculation into fresh medium. In continuous-culture experiments, conversion of lacZ substrate was demonstrated microscopically in individual cells by epifluorescence microscopy.

94 CHANNELING OF IMP INTO GUANYLATE SYNTHESIS IN THE GROWTH PROGRAM OF HEPATOMA 3924A CELLS

The purpose was to elucidate the relationship of IMP utilization for de novo guanylate and adenylate synthesis during the display of the proliferative program of hepatoma 3924A cells. Purine compounds in the cells were labeled with [14C] formate or L- [3-14C]serine in a serine-free assay medium, hydrolyzed to purine bases, separated and counted. Under these assay conditions labeling of adenine or guanine was proportionate with incubation time and with number of cells. During the transition of hepatoma cells from plateau phase into logarithmic proliferation the concentration of PRPP increased 14-fold, followed by an 8-fold rise in purine de novo synthesis. In plateau phase cells predominantly adenylates were produced. After replating the resting cells there was a sharp increase in the relative labeling of guanine with a concurrent marked decrease in that of adenine in the late lag phase. The subsequent decline of the relative labeling of guanine was accompanied with the restoration in that of adenine with the increase in the cell density. The striking redirection in the distribution of label from IMP utilization to the preferential synthesis of guanylates during the expression of the biochemical proliferative program of cancer cells underlines the potential significance of this pathway as a target of chemotherapy. (Supported by Outstanding Investigator Grant CA-42510 to G.W.)

Distribution of cysteine conjugate beta-lyase in gastrointestinal bacteria and in the environment.

A cysteine conjugate beta-lyase was detected in 24 of 43 gastrointestinal bacteria tested, and from mixed populations of bacteria obtained from lake, river and soil samples. These bacterial cysteine conjugate beta-lyases catalyse the cleavage of the thioether linkage of both an S-alkyl- and an S-aryl-linked cysteine conjugate (2-S-cysteinyl-N-isopropylacetanilide (cysteine conjugate of propachlor) and S-(2-benzothiazolylcysteine), respectively). A cysteine conjugate beta-lyase was isolated from Eubacterium limosum at levels at least 16-fold greater than those from any other gastrointestinal bacterial tested. This enzymic activity was present from the late lag phase through the stationary phase of growth of the bacteria. Maximum activity occurred in the mid log phase. A cysteine conjugate beta-lyase isolated from animal and plant tissues cleaved only the S-aryl-linked cysteine conjugate (S-(2-benzothiazolyl)cysteine) and generally had less enzymic activity than enzymes isolated from the bacteria. Glutathione-S-transferase activity was not detected in the 43 gastrointestinal bacteria tested, except for a low level (0.6 nmol/min per mg) of activity in Escherichia coli. No S-methyl transferase activity was detected in the gastrointestinal bacteria or mixed populations of bacteria tested.

Variations in ribulose 1,5-bisphosphate carboxylase protein levels, activities and subcellular distribution during photoautotrophic batch culture of Chlorogloeopsis fritschii

Ribulose 1,5-bisphosphate (RuBP) carboxylase is present in the cytoplasm and carboxysomes (polyhedral bodies) of the cyanobacterium Chlorogloeopsis fritschii. In vitro enzyme activities have been measured throughout photoautotrophic batch culture, together with RuBP carboxylase protein concentrations, determined by rocket immunoelectrophoresis. Enzyme activities and protein levels in the cytoplasmic and carboxysomal fractions varied in an apparently inverse manner during growth. The RuBP carboxylase activities per unit enzyme protein were maximal in late lag phase/early exponential phase for both cellular enzyme pools. Both rates per unit enzyme protein declined during exponential phase, cytoplasmic enzyme activity remaining consistently higher than that of the carboxysomal enzyme. Activities per unit cytoplasmic and carboxysomal enzyme protein showed very low, similar rates in late stationary phase and death phase. Dialysis experiments indicated that such changes were not due to interference in activity assays by soluble endogenous effectors. Major shifts in the subcellular distribution of RuBP carboxylase protein were found versus culture age, enzyme protein levels being predominantly carboxysomal in lag phase, mainly soluble in exponential phase and then mainly carboxysomal again in stationary/death phase. The data are discussed in terms of carboxysome function and the question of control of RuBP carboxylase synthesis in cyanobacteria.

Inherent limitations to the problem of reducing the lysine microbiological assay time.

A kinetic approach is proposed to shorten the microbiological assay time for the determination of unbound L-lysine. The present lysine bacterial assay takes from 16 to 24 h using Pediococcus cerevisiae P-60 ATCC 8042 (formerly Leuconostoc mesenteroides P-60 ATCC 8042) and uses a medium in which lysine is the limiting substance. Measurements of the final cell concentration are linearly correlated with the initial concentration of lysine, S, to provide an indirect estimate of S. We propose to understand the limitations inherent to the reduction of the assay time to 4 h by focusing in our analysis on the bacterial late lag or early growth transient phases, rather than the stationary phase of growth. Generally, the Monod equation is expected to describe a hyperbolically increasing correlation between the bacterial specific growth rate at about 2-4 h and the initial lysine concentration. A hyperbolic correlation is obtained by 3 h, but the lysine region of interest falls in the saturated portion of the curve. Discriminations between different initial lysine levels are therefore difficult with this nearly flat curve. On the other hand, when the initial inoculum level is lowered, so that substrate inhibition becomes effective, a correlation with a large negative slope is obtained by 4 h. Limitations to using absorbance measurements for the rapid assay turn up in a lack of reproducibility and, hence, a large variance associated with the measurements. Alternative microbial measuring techniques, such as impedance methods, need to be examined in order to reduce that large variance.