Use of Stochastic Models To Assess the Effect of Environmental Factors on Microbial Growth

We present a novel application of a stochastic ecological model to the study and analysis of microbial growth dynamics as influenced by environmental conditions in an extensive experimental data set. The model proved to be useful in bridging the gap between theoretical ideas in ecology and an applied problem in microbiology. The data consisted of recorded growth curves of Escherichia coli grown in triplicate in a base medium with all 32 possible combinations of five supplements: glucose, NH₄Cl, HCl, EDTA, and NaCl. The potential complexity of 2⁵ experimental treatments and their effects was reduced to 2² as just the metal chelator EDTA, the presumed osmotic pressure imposed by NaCl, and the interaction between these two factors were enough to explain the variability seen in the data. The statistical analysis showed that the positive and negative effects of the five chemical supplements and their combinations were directly translated into an increase or decrease in time required to attain stationary phase and the population size at which the stationary phase started. The stochastic ecological model proved to be useful, as it effectively explained and summarized the uncertainty seen in the recorded growth curves. Our findings have broad implications for both basic and applied research and illustrate how stochastic mathematical modeling coupled with rigorous statistical methods can be of great assistance in understanding basic processes in microbial ecology.     

Spatial and short-term variability of larval,post-larval and macrobenthic assemblages associated with subtidal kelp forest ecosystems in Central Chile

Identifying patterns of spatial and temporal variability in the composition of communities associated with kelp forests is critical to understand the functioning of this productive, yet vulnerable ecosystem. We used a suite of sampling methods (light attraction and airlift devices) to evaluate the variability of larval, post-larval and macrobenthic assemblages associated with kelp forests (Lessonia trabeculata) in Central Chile (30° to 33°S). Pelagic collections identified two assemblages: early-life stages and emerging macrobenthos, with the later contributing three quarters to the total abundance regardless of the source of illumination (permanent or flashing). Field experiments showed that moon phases affected the structure and composition of the samples. Surveys carried out during new moon showed the highest abundances and taxonomic richness of emergent assemblages. However, species composition varied in both assemblages depending on the moon phase. Although the pelagic assemblages collected at sites with contrasting upwelling intensity did not show differences in community structure, differences in composition were evident for early-life stages. The relationship between pelagic and benthic collections indicated that four decapod crustaceans were represented at both larval and early juvenile stages; however, only the high abundances and densities of Paraxanthus barbiger allowed for estimations of benthic-pelagic coupling. For this species, larval abundances and benthic juvenile densities demonstrated contrasting local and regional patterns, suggesting a decoupling between pelagic and benthic environments. These findings highlight the differential variability in smaller components of kelp forests, but also suggest that post-settlement processes may be driving biological interactions through these highly productive and complex environments.     

Quantification of Expression of Staphylococcus epidermidis Housekeeping Genes with Taqman Quantitative PCR during In Vitro Growth and under Different Conditions

The aims of the present study were (i) to develop and test a sensitive and reproducible method for the study of gene expression in staphylococci and (ii) to study the expression of five housekeeping genes which are involved in nucleic acid metabolism (gmk, guanylate kinase; the dihydrofolate reductase [DHFR] gene), glucose metabolism (tpi, triosephosphate isomerase), and protein metabolism (the 16S rRNA gene; hsp-60, heat-shock protein 60) during in vitro exponential and stationary growth. A modified method for instant mRNA isolation was combined with gene quantification via Taqman real-time quantitative PCR. The detection limit of our method was 10 copies of RNA. The average intersample variability was 16%. A 10-fold increase in the expression of the hsp-60 gene was induced by exposure to a 10 degrees C heat shock (37 to 47 degrees C) for 10 min. During in vitro growth, the expression of all five housekeeping genes showed rapid up-regulation after inoculation of the bacteria in brain heart infusion medum and started to decline during the mid-exponential-growth phase. Maximal gene expression was 110- to 300-fold higher than gene expression during stationary phase. This indicates that housekeeping metabolism is a very dynamic process that is extremely capable of adapting to different growth conditions. Expression of the 16S rRNA gene decreases significantly earlier than that of other housekeeping genes. This confirms earlier findings for Escherichia coli that a decline in bacterial ribosomal content (measured by 16S rRNA gene expression) precedes the decline in protein synthesis (measured by mRNA expression).     

Substrate availability in phenanthrene biodegradation: Transfer mechanism and influence on metabolism

 The mechanism of phenanthrene transfer to the bacteria during biodegradation by a Pseudomonas strain was investigated using a sensitive respirometric technique (Sapromat equipment) allowing the quasi-continuous acquisition of data on oxygen consumption. Several systems of phenanthrene supply, crystalline solid and solutions in non-water-miscible solvents (silicone oil and 2,2,4,4,6,8,8-heptamethylnonane) were studied. In all cases, analysis of the kinetics of oxygen consumption demonstrated an initial phase of exponential growth with the same specific growth rate. In order to analyze the second phase of growth and phenanthrene degradation, a study of the kinetics of phenanthrene transfer to the aqueous phase was conducted by direct experimentation, with the crystal and silicone oil systems, in abiotic conditions. The data allowed the validation of a model based on phase-transfer laws, describing the variations, with substrate concentrations, of rates of phenanthrene transfer to the aqueous phase. Analysis of the biodegradation curves then showed that exponential growth ended in all cases when the rates of phenanthrene consumption reached the maximal transfer rates. Thereafter, the biodegradation rates closely obeyed, for all systems, the transfer rate values given by the model. These results unambiguously demonstrated that, in the present case, phenanthrene biodegradation required prior transfer to the aqueous phase. With the silicone oil system, which allowed high transfer and biodegradation rates, phenanthrene was directed towards higher metabolite production and lower mineralization, as shown by oxygen consumption and carbon balance determinations. Received: 30 November 1994/Accepted: 11 January 1995     

Control of the selectivity of butyric acid production and improvement of fermentation performance withClostridium tyrobutyricum

Summary The parameters that control fermentation performance of butyrate production have been studied with a selected strain ofClostridium tyrobutyricum. Fed-batch supply of glucose increased productivity for butyrate. The ratio of butyrate to total acids was strongly influenced by the growth rate of the bacteria, acetate being produced along with butyrate at higher growth rates. In glucose-limited, fed-batch cultures, initially produced acetate was re-utilized, resulting in exclusive production of butyrate. In cultures with non-limiting glucose feeding, the butyrate concentration reached 42.5 g·1^–1 with a selectivity of 0.90, a productivity of 0.82 g·^–1 per hour and a yield of 0.36 g·g^–1 The effects of the mode of supply of glucose on the production of butyrate and acetate are discussed in relation with the energy requirements for cell growth.     

Industrial optimization of acetone-butanol fermentation: a study of the utilization of Jerusalem artichokes

Summary Acetone-butanol fermentation of the Jerusalem artichoke has been studied as a case for systematic investigation of the industrial optimization of both strain selection and fermentation operation. Hydrolysis of the inulinic oligofructans of the substrate was found necessary for optimal performance but could be achieved with a selected strain using a moderate amount of inulinase added at the beginning of the fermentation. Apart from ammonia, no nutritional supplementation of the medium was found necessary. The marked influence of pH in the fermentation performance prompted a detailed search for a method of controlling pH during fermentation. With an optimized procedure, solvent production of 23–24 g/l were obtained in 36 h. Detailed fermentation balances are presented. An industrial process for ABE production from Jerusalem artichoke or sugar beet has been defined and tested in the pilot plant.     

Species- and tissue-dependent effects of NO and cyclic GMP on cardiac ion channels

Biochemical studies have established the presence of a NO pathway in the heart, including sources of NO and various effectors. Several cardiac ion channels have been shown to be modified by NO, such as L-type Ca(2+), ATP-sensitive K(+), and pacemaker f-channels. Some of these effects are mediated by cGMP, through the activity of three main proteins: the cGMP-dependent protein kinase (PKG), the cGMP-stimulated phosphodiesterase (PDE2) and the cGMP-inhibited PDE (PDE3). Other effects appear independent of cGMP, as for instance the NO modulation of the ryanodine receptor-Ca(2+) channel. In the case of the cardiac L-type Ca(2+) channel current (I(Ca,L)), both cGMP-dependent and cGMP-independent effects have been reported, with important tissue and species specificity. For instance, in rabbit sinoatrial myocytes, NO inhibits the beta-adrenergic stimulation of I(Ca,L) through activation of PDE2. In cat and human atrial myocytes, NO potentiates the cAMP-dependent stimulation of I(Ca,L) through inhibition of PDE3. In rabbit atrial myocytes, NO enhances I(Ca,L) in a cAMP-independent manner through the activation of PKG. In ventricular myocytes, NO exerts opposite effects on I(Ca,L): an inhibition mediated by PKG in mammalian myocytes but by PDE2 in frog myocytes; a stimulation attributed to PDE3 inhibition in frog ventricular myocytes but to a direct effect of NO in ferret ventricular myocytes. Finally, NO can also regulate cardiac ion channels by a direct action on G-proteins and adenylyl cyclase.     

Negative feedback exerted by cAMP-dependent protein kinase and cAMP phosphodiesterase on subsarcolemmal cAMP signals in intact cardiac myocytes: an in vivo study using adenovirus-mediated expression of CNG channels

Intracardiac cAMP levels are modulated by hormones and neuromediators with specific effects on contractility and metabolism. To understand how the same second messenger conveys different information, mutants of the rat olfactory cyclic nucleotide-gated (CNG) channel alpha-subunit CNGA2, encoded into adenoviruses, were used to monitor cAMP in adult rat ventricular myocytes. CNGA2 was not found in native myocytes but was strongly expressed in infected cells. In whole cell patch-clamp experiments, the forskolin analogue L-858051 (L-85) elicited a non-selective, Mg2+ -sensitive current observed only in infected cells, which was thus identified as the CNG current (ICNG). The beta-adrenergic agonist isoprenaline (ISO) also activated ICNG, although the maximal efficiency was approximately 5 times lower than with L-85. However, ISO and L-85 exerted a similar maximal increase of the L-type Ca2+ current. The use of a CNGA2 mutant with a higher sensitivity for cAMP indicated that this difference is caused by the activation of a localized fraction of CNG channels by ISO. cAMP-dependent protein kinase (PKA) blockade with H89 or PKI, or phosphodiesterase (PDE) inhibition with IBMX, dramatically potentiated ISO- and L-85-stimulated ICNG. A similar potentiation of beta-adrenergic stimulation occurred when PDE4 was blocked, whereas PDE3 inhibition had a smaller effect (by 2-fold). ISO and L-85 increased total PDE3 and PDE4 activities in cardiomyocytes, although this effect was insensitive to H89. However, in the presence of IBMX, H89 had no effect on ISO stimulation of ICNG. This study demonstrates that subsarcolemmal cAMP levels are dynamically regulated by a negative feedback involving PKA stimulation of subsarcolemmal cAMP-PDE.     

Purification and Properties of the Constituents of the Nitrogenase Complex from Clostridium pasteurianum

A new procedure for a rapid and extensive purification of the FeMo protein and the Fe protein of the nitrogenase complex from Clostridium pasteurianum is described. Specific activities of 345 and 460 nmoles of N(2) reduced per mg of protein per min for the FeMo protein and for the Fe protein, respectively, have been obtained. Preparations of the FeMo protein contained 0.96 atom of molybdenum and 15 atoms of iron per molecule, whereas those of the Fe protein contained 2.86 atoms of iron per molecule. Experiments suggest that a definite association of two Fe proteins and one FeMo protein is functional in the active enzyme complex. No individual role could be ascribed to either of the two proteins, but the fact that hydrogenase inhibits N(2) fixation but not the reductant-dependent adenosine triphosphate hydrolysis supports the idea that there are two distinct sites on nitrogenase, one concerned with N(2) activation and the other with activated electron transport.