Improved polymerase ribozyme efficiency on hydrophobic assemblies

During an early step in the evolution of life, RNA served both as genome and as catalyst, according to the RNA world hypothesis. For self-replication, the RNA organisms must have contained an RNA that catalyzes RNA polymerization. As a first step toward recapitulating an RNA world in the laboratory, a polymerase ribozyme was generated previously by in vitro evolution and design. However, the efficiency of this ribozyme is about 100-fold too low for self-replication because of a low affinity of the ribozyme to its primer/template substrate. To improve the substrate interactions by colocalizing ribozyme and substrate on micelles, we attached hydrophobic anchors to both RNAs. We show here that the hydrophobic anchors led to aggregates with the expected size of the corresponding micelles. The micelle formation increased the polymerization yield of full-length products by 3- to 20-fold, depending on substrates and reaction conditions. With the best-characterized substrate, the improvement in polymerization efficiency was primarily due to reduced sequence-specific stalling on partially extended substrates. We discuss how, during the origin of life, micellar ribozyme aggregates could have acted as precursors to membrane-encapsulated life forms.     

The ideal free distribution and bacterial growth on two substrates

A population dynamical model describing growth of bacteria on two substrates is analyzed. The model assumes that bacteria choose substrates in order to maximize their per capita population growth rate. For batch bacterial growth, the model predicts that as the concentration of the preferred substrate decreases there will be a time at which both substrates provide bacteria with the same fitness and both substrates will be used simultaneously thereafter. Preferences for either substrate are computed as a function of substrate concentrations. The predicted time of switching is calculated for some experimental data given in the literature and it is shown that the fit between predicted and observed values is good. For bacterial growth in the chemostat, the model predicts that at low dilution rates bacteria should feed on both substrates while at higher dilution rates bacteria should feed on the preferred substrate only. Adaptive use of substrates permits bacteria to survive in the chemostat at higher dilution rates when compared with non-adaptive bacteria.     

Bacterial Growth on Surfaces: Automated Image Analysis for Quantification of Growth Rate-Related Parameters

A fast routine method for estimating bacterial cell growth rates by using the metachromatic dye acridine orange is described. The method allows simultaneous estimates of cellular RNA and DNA contents of single cells. Acridine orange staining can be used as a nonspecific supplement to quantitative species-specific hybridizations with fluorescence-labelled ribosomal probes to estimate the single-cell concentration of RNA. By automated analysis of digitized images of stained cells, we determined four independent growth rate-related parameters: cellular RNA and DNA contents, cell volume, and the frequency of dividing cells in a cell population. These parameters were used to compare physiological states of liquid-suspended and surface-growing Pseudomonas putida KT2442 in chemostat cultures. The major finding is that the correlation between substrate availability and cellular growth rate found for the free-living cells was not observed for the surface-bound cells; in contrast, the data indicate an almost constant growth rate for attached cells which was independent of the dilution rate in the chemostat.     

Analyses of microbial activity in biomass-recycle reactors using denaturing gradient gel electrophoresis of 16S rDNA and 16S rRNA PCR products

The relationship between mixed microbial community structure and physiology when grown under substrate-limited conditions was investigated using continuous-flow bioreactors with 100% biomass recycle. Community structure was analyzed by denaturing gradient gel electrophoresis (DGGE) of the PCR and RT-PCR amplified V3 region of 16S rDNA and 16S rRNA templates, respectively. Comparisons were made of communities exposed to different types of transient conditions (e.g., long- and short-term starvation, increasing nutrients). With progressively more stringent substrate limitation over time, the specific content of community RNA declined by more than 10-fold and closely followed the decline in specific growth rate. In contrast, the DNA content was variable (up to 3-fold differences) and did not follow the same trend. Cluster analysis of the presence or absence of individual bands indicated that the fingerprints generated by the two templates were different, and community response was first observed in the rRNA fraction. However, both the rDNA and rRNA fingerprints provided a picture of temporal population dynamics. Dice similarity coefficients gave a quantitative measure of the differences and changes between the communities. In comparison, standard cultivation techniques yielded only a quarter of the phylotypes detected by DGGE, but included the most dominant population based on rRNA. Nucleotide-sequence analyses of the almost complete 16S rRNA genes of these isolates place them in the same group of organisms that is typically cultivated from environmental samples: alpha, beta, and gamma Proteobacteria and the high GC and the low GC Gram-positive divisions.     

A flow cytometry analysis of batch and continuous culture transient diauxic growth in Hansenula polymorpha

A flow cytometry analysis and in vitro enzyme activity study is carried out on the methylotrophic yeast, Hansenula polymorpha, during both (a) batch growth and (b) continuous cultures subjected to single perturbations in either system dilution rate or influent carbon substrate composition. Flow cytometry of yeasts growing diauxically on a glucose: methanol mixture during exponential growth, exhibit DNA and RNA distributions indicative of the S-synthesis-phase of the cell cycle. Cells at the stationary growth stage exhibit DNA and RNA distributions that indicate one portion of the population in the G ₀/G₁ resting phase and another in the M-mitosis-phase.Yeast cells grown at a steady-state of D=0.2 h¹, then shifted to D=0.35 h^–1, at a constant influent substrate mixture, are also examined with both flow cytometry and in vitro enzyme assays. Distributions of DNA, RNA, and total protein at either steady state and during the shift between dilution rates did not resemble any observed in batch culture. Flow cytometry indicates significant changes in cell composition within 20 min of the imposed dilution rate shift. In vitro enzyme assays show a response time in decreasing methanol oxidase activity of 2.5–3 h upon a dilution rate shift-up, while hexokinase activity increases to its steady-state level in less than 3 h. Similar cell compositional changes are reported for shifts in influent substrate methanol: glucose ratio at a constant dilution rate of D=0.35 h ^–1. Results suggest that an unsteady-state regime, oscillating between conditions that promote maximum enzyme activity of either glucose- or methanol-metabolizing enzymes, may allow simultaneous enhanced time-averaged production of both sets of enzymes.     

Regulation of RNA synthesis in plant cell culture: delayed synthesis of ribosomal RNA during transition from the stationary phase to active growth

Ribosomal RNA synthesis was studied during the early phases of growth activation in a cell suspension culture derived from peanut (Arachis hypogaea, L.) cotyledon. Upon dilution from stationary phase, these cells show a characteristic lag of 3 days before the commencement of cell division. An analysis of the nature of RNA synthesized during this early period of growth showed that the cells obtained immediately upon dilution from stationary phase synthesize primarily messenger RNA and essentially no ribosomal RNA. The synthesis of ribosomal RNA is delayed for about 24 hr after which it rises sharply resulting in a 2- to 3-fold accumulation of ribosomal RNA per cell during the subsequent 24-hr period. Both the messenger RNA and the ribosomal RNA were characterized by their cellular localization; by sucrose and CsCl gradient analyses, and by the determination of their base ratios.It would appear that a major facet of the lag phase in the cell growth is the diversion of a significant part of the RNA biosynthetic apparatus from the synthesis of messenger RNA to that of ribosomal RNA.     

Continuous In Vitro Evolution of Ribozymes That Operate Under Conditions of Extreme pH

Continuous in vitro evolution methods were used to study the behavior of an evolving population of RNA ligase ribozymes in response to selection pressures involving conditions of extreme pH. The starting population consisted of randomized variants of a ribozyme that had been optimized for activity at pH 8.5. The ribozymes were subjected to repeated rounds of selective amplification under progressively more acidic or more alkaline conditions. The two final evolved populations of ribozymes were able to operate at either pH 5.8 or pH 9.8, respectively. Representative individuals from the two final populations were isolated and characterized. The low-pH ribozyme exhibited a 10-fold increase in catalytic rate at pH 5.8 compared to the starting molecule. The high-pH ribozyme retained its structural integrity and activity at pH 9.8, whereas the starting molecule was denatured under this condition. These findings demonstrate that a population of functional macromolecules can adapt to stringent environmental conditions through the acquisition of relatively few mutations. The results establish continuous in vitro evolution as a useful model system for exploring the evolution of enzymatic function in extreme environments. Present address (Henriette Kühne): Cardinal Health, 2950 Trade Place, San Diego, CA 92126, USA     

Transitional states in a chemostat culture of Candida utilis

Transient states of the chemostat Candida utilis 1668-3-37 culture were studied when its growth was limited by ethanol and an abrupt acidification of the medium from pH 5.0 to 2.2 was done or when the dilution rate was rapidly changed from D = 0.1 to 0.3 h-1 and back to 0.07 h-1. The pH shock was found to cause stronger oscillations in a number of parameters (the weight of dry biomass, the content of residual ethanol, the content of RNA in the cells) than a change in the dilution rate. In the latter case the population density changed more smoothly than the content of RNA did. DNA content remained at one and the same level in all of the experiments. All of the oscillations were observed only in the first generation after a shock; there upon, the culture remained for a long time (7 to 10 generations) in a very stable state typical of chemostat cultures. The oscillations induced by the unfavourable pH of the medium were compared with those caused by an abrupt change in the dilution rate. The pH shock brought about multiple damping oscillations of the parameters whereas a change in the dilution rate resulted, most often, in a merely one oscillation.     

Effect of variable nutrient supply rates on the RNA level of a heterotrophic bacterial strain

The heterotrophic bacterial strain HIS 53 was grown in a continuous culture under chemostat conditions and at sinusoidal or stepwise variations of the dilution rate; aspartate, ammonium, and phosphate were the growth-limiting nutrients. Within a specific nutrient limitation the growth yield was constant and independent of the applied environmental conditions. Compared with the reference chemostat culture, sinusoidal variations of the dilution rate increased the cellular RNA level by 19%–53% dependent on the growth limitation; stepwise variations caused an increase of the RNA level by 28%–41%. It was hypothesized that under the variable environmental conditions in the natural habitat the physiological potential of the organism is enhanced by some such increase of the cellular RNA level. As a consequence these increased RNA levels influence the competition between heterotrophic bacteria and, as a result, also the composition of the population of heterotrophic bacteria.     

Evolution of structure in RNA

Evidence is presented that structures formed by RNA and by RNA in association with protein have evolved from simpler structures by successive unions among them. The progressively more complex molecular structures have conferred selective advantage in evolution by progressively enhancing the specificities of the biochemical reactions. Before each union, the RNAs which joined at the time of union belonged to separate reproducing species. The record of unions in RNA therefore reflects unions among species in the biosphere, tracing the evolution of life from quite simple reproducing molecules up to well developed organisms.     

Ferrowax microvalves for fully automated serial dilution on centrifugal microfluidic platforms

We herein describe a centrifugal microfluidic system to accomplish a fully automated serial dilution. The liquid flow on the disc was regulated by utilizing ferrowax microvalves systematically integrated into the channels within specially designed metering structures. By opening the differently positioned microvalves through irradiation of IR laser to allow metering, the same amount of diluent was serially eluted to the dilution chamber from the same diluent chamber. After dilution, the diluted samples were automatically delivered to the respective final product chambers by appropriately opening or closing the microvalves in the connecting channels, followed by rotating the disc. Based on this unique design principle, six consecutive two-fold and 10-fold dilutions were successfully achieved, yielding excellent accuracy in a wide dynamic range up to six orders of magnitude. Very importantly, the overall serial dilution process, including the diluent addition, mixing, and product transfer steps, was completed very rapidly within 5 min, due to the minimized procedures enabled by the automated actuation of the ferrowax microvalves at the rationally designed positions. We expect our centrifugal microfluidic system would serve as a powerful elemental tool to realize fully automated diagnostic microsystems involving the serial dilution process.     

Regulation of cellulase synthesis in batch and continuous cultures of Clostridium thermocellum

Regulation of cell-specific cellulase synthesis (expressed in milligrams of cellulase per gram [dry weight] of cells) by Clostridium thermocellum was investigated using an enzyme-linked immunosorbent assay protocol based on antibody raised against a peptide sequence from the scaffoldin protein of the cellulosome (Zhang and Lynd, Anal. Chem. 75:219-227, 2003). The cellulase synthesis in Avicel-grown batch cultures was ninefold greater than that in cellobiose-grown batch cultures. In substrate-limited continuous cultures, however, the cellulase synthesis with Avicel-grown cultures was 1.3- to 2.4-fold greater than that in cellobiose-grown cultures, depending on the dilution rate. The differences between the cellulase yields observed during carbon-limited growth on cellulose and the cellulase yields observed during carbon-limited growth on cellobiose at the same dilution rate suggest that hydrolysis products other than cellobiose affect cellulase synthesis during growth on cellulose and/or that the presence of insoluble cellulose triggers an increase in cellulase synthesis. Continuous cellobiose-grown cultures maintained either at high dilution rates or with a high feed substrate concentration exhibited decreased cellulase synthesis; there was a large (sevenfold) decrease between 0 and 0.2 g of cellobiose per liter, and there was a much more gradual further decrease for cellobiose concentrations >0.2 g/liter. Several factors suggest that cellulase synthesis in C. thermocellum is regulated by catabolite repression. These factors include: (i) substantially higher cellulase yields observed during batch growth on Avicel than during batch growth on cellobiose, (ii) a strong negative correlation between the cellobiose concentration and the cellulase yield in continuous cultures with varied dilution rates at a constant feed substrate concentration and also with varied feed substrate concentrations at a constant dilution rate, and (iii) the presence of sequences corresponding to key elements of catabolite repression systems in the C. thermocellum genome.     

Mixed Continuous Cultures of Polyvinyl Alcohol-Utilizing Symbionts Pseudomonas putida VM15A and Pseudomonas sp. Strain VM15C

Stable mixed continuous cultures of Pseudomonas sp. strain VM15C and Pseudomonas putida VM15A, the former of which produced a polyvinyl alcohol (PVA)-degrading enzyme and the latter of which produced an essential growth factor for PVA utilization by strain VM15C, were established with PVA as the sole source of carbon and energy with chemostat cultivation. A high extent of PVA degradation was achieved at dilution rates of less than 0.030/h. The predominant strain in the cultures was the primary metabolizer of PVA, strain VM15C. The growth supporter, strain VM15A, existed as a minor population, although its population was maintained at an almost constant level throughout a dilution region in which the VM15C population decreased markedly as the dilution rate was raised. A crude growth factor which was prepared from a culture supernatant of strain VM15A and increased the specific growth rate of strain VM15C with PVA in an axenic batch culture was also effective for enhancing the VM15C population and PVA degradation in the mixed continuous culture at a high dilution rate (0.064/h). This indicated that the growth-limiting substrate for strain VM15C in the mixed continuous culture is the growth factor produced by strain VM15A.     

Acquisition of novel catalytic activity by the M1 RNA ribozyme: the cost of molecular adaptation.

The ribonucleoprotein RNase P is a critical component of metabolism in all known organisms. In Escherichia coli, RNase P processes a vast array of substrates, including precursor-tRNAs and precursor 4. 5S RNA. In order to understand how such catalytic versatility is achieved and how novel catalytic activity can be acquired, we evolve the M1 RNA ribozyme (the catalytic component of E. coli RNase P) in vitro for cleavage of a DNA substrate. In so doing, we probe the consequences of enhancing catalytic activity on a novel substrate and investigate the cost this versatile enzyme pays for molecular adaptation. A total of 25 generations of in vitro evolution yield a population showing more than a 1000-fold increase in DNA substrate cleavage efficiency (kcat/KM) relative to wild-type M1 RNA. This enhancement is accompanied by a significant reduction in the ability of evolved ribozymes to process the ptRNA class of substrates but also a contrasting increase in activity on the p4.5S RNA class of substrates. This change in the catalytic versatility of the evolved ribozymes suggests that the acquired activity comes at the cost of substrate versatility, and indicates that E. coli RNase P catalytic flexibility is maintained in vivo by selection for the processing of multiple substrates. M1 RNA derivatives enhance cleavage of the DNA substrate by accelerating the catalytic step (kcat) of DNA cleavage, although overall processing efficiency is offset by reduced substrate binding. The enhanced ability to cleave a DNA substrate cannot be readily traced to any of the predominant mutations found in the evolved population, and must instead be due to multiple sequence changes dispersed throughout the molecule. This conclusion underscores the difficulty of correlating observed mutations with changes in catalytic behavior, even in simple biological catalysts for which three-dimensional models are available.     

Factors affecting the formation of polysaccharide depolymerase and glycoside hydrolyase enzymes by Butyrivibrio fibrisolvens NCDO 2249

Specific activities of hemicellulose-degrading polysaccharide depolymerase and glycoside hydrolase enzymes were measured in batch and continuous cultures of Butyrivibrio fibrisolvens NCDO 2249 grown on cellobiose or a hemicellulosic carbohydrate. Enzyme activities were influenced by the growth substrate and by the rate and stage of growth of the micro-organism. In cellobiose batch cultures specific activities were maximal as the growth rate declined and in the initial stages of the stationary phase. The growth substrate did not affect the range of glycoside hydrolases formed, although specific activities were substrate-dependent, with activity increases (up to 200-fold) occurring in enzymes essential for effective substrate utilization. Appreciable xylanase activity was present only in xylan-grown cultures. The substrate effects were also evident in chemostat cultures. The activity response of the nine enzymes monitored to growth rate changes differed in that while the activity of some enzymes, including xylanase, declined at high dilution rates the activities of others were not growth rate-dependent and were maintained over the range of dilution rates examined. Exocellular activities were detected only in spent media from cultures grown with a polymeric (hemicellulosic) carbohydrate.     

Physiological state of a microbial community in a biomass recycle reactor

The transition in physiological state was investigated between a carbon-limited chemostat population and microbes growing very slowly in a biomass recycle reactor. The mixed microbial population was metabolizing a mixture of biopolymers and linear alkylbenzene sulfonate, formulated to represent the organic load in graywater. Biomass increased 30-fold during the first 14 days after a shift from chemostat to biomass recycle mode. The ratios of ATP and RNA to cell protein decreased over the first days but then remained constant. The specific rate of CO₂ production by microbes in the reactor decreased 6-fold within 24 h after the shift, and respiratory potentials declined 2–3 fold during the first 7 days. Whereas chemostat cultures used equal proportions of organic carbon substrate for catabolism and anabolism, the proportion of organic substrate oxidized to CO₂ rose from 62 to 82% over the first 8 days in a biomass recycle reactor, and eventually reached 100% as this reactor population exhibited no net growth. Biomass recycle populations removed from the system and subjected to a nutritional shift-up did not immediately initiate exponential growth. The physiological state of cells in the biomass recycle reactor may be distinct from those grown in batch or continuous culture, or from starved cells. Received 02 June 1997/ Accepted in revised form 20 February 1998     

Batch- and Continuous-Culture Transients for Two Substrate Systems

Batch growth of Escherichia coli in the presence of equal initial concentrations of glucose and a secondary substrate (xylose) is characterized by sequential utilization of the substrates, whereas continuous-culture systems with equal concentrations of the two substrates in the feed are characterized by complete utilization of both substrates at both high and low dilution rates. Such systems at steady state at a low dilution rate, when suddenly shifted to a higher dilution rate, experience a transient drop in population density accompanied by accumulation of the secondary substrate but virtually no accumulation of glucose. Systems at steady state with 200 mg of glucose per liter were found to undergo a transient population decrease and eventual recovery when switched to feed containing 200 mg of a secondary substrate per liter.     

Self-Cycling Fermentation Applied to Acinetobacter calcoaceticus RAG-1

The self-cycling fermentation (SCF) technique was applied to a culture of Acinetobacter calcoaceticus RAG-1. This method was shown to result in synchronization of the cells, achieving a 77% improvement in cell synchrony over that of the batch case. Cellular occurrences, averaged out by asynchronous batch cultures, were magnified by the temporal alignment of metabolic events brought about by the synchronization associated with SCFs. The cell population doubled only once per cycle, thus establishing an equality between cycle time and doubling time. Parameters of interest were biomass concentration, total bioemulsifier (emulsan) production, cycle time, and residual carbon concentration. Cycle-to-cycle variation of these parameters was, in most cases, insignificant. Repeatability of doubling time estimates (based on 95% confidence intervals) was roughly 7 to 10 times better between cycles in an SCF than between batch replicates. The carbon substrate was completely utilized in all cases in which it was measured, giving this technique an advantage over chemostat-type fermentations. The dissolved-oxygen profiles monitored throughout a cycle were found to be repeatable. A characteristic shape, which can be related to the growth of the organism, was associated with each carbon source. The specific emulsan productivity of SCFs was found to be approximately 50 times greater than that of the batch process and 2 to 9 times greater than that of the chemostat, depending on the dilution rate considered. With respect to specific emulsan production, a 25-fold improvement over that in an immobilized cell system recently introduced was obtained. Thus, SCFs are a viable alternative to established fermentation techniques.     

The dynamics of single-substrate continuous cultures: the role of ribosomes

When a chemostat is perturbed from its steady state, it displays complex dynamics. For instance, if the identity of the growth-limiting substrate is switched abruptly, the substrate concentration and cell density undergo a pronounced excursion from the steady state that can last several days. These dynamics occur because certain physiological variables respond slowly. In the literature, several physiological variables have been postulated as potential sources of the slow response. These include transport enzymes, biosynthetic enzymes, and ribosomes. We have been addressing this problem by systematically exploring the role of these variables. In previous work Shoemaker et al. (J. Theor. Biol., 222 (2003) 307-322), we studied the role of transport enzymes, and we showed that transients starting from low transport enzyme levels could be quantitatively captured by a model taking due account of transport enzyme synthesis. However, there is some experimental data indicating that slow responses occur even if the initial enzyme levels are high. Here, we analyse this data to show that in these cases, the sluggish response is most probably due to slow adjustment of the ribosome levels. To test this hypothesis, we extend our previous model by accounting for the evolution of both the transport enzyme and the ribosomes. Based on a kinetic analysis of the data in the literature, we assume that the specific protein synthesis rate is proportional to the ribosome level, and the specific ribosome synthesis rate is autocatalytic. Simulations of the model show remarkable agreement with experimentally observed steady states and the transients. Specifically, the model predictions are in good agreement with (1) the steady-state profiles of the cell density, substrate concentration, RNA, proteins, and transport enzymes, (2) the instantaneous specific substrate uptake, growth, and respiration rates in response to a continuous-to-batch shift, and (3) the transient profiles of the cell density, substrate concentration, and RNA in response to feed switches and dilution rate shifts. Time-scale analysis of the model reveals that every transient response is a combination of two fundamental (and simpler) dynamics, namely, substrate-sufficient batch dynamics and cell-sufficient fed-batch dynamics. We obtain further insight into the transient response by analysing the equations describing these fundamental dynamics. The analysis reveals that in feed switches or dilution rate shift-ups, the transport enzyme reaches a maximum before RNA achieves its maximum, and in dilution rate shift-downs the cell density reaches a maximum before RNA achieves a minimum.