The dynamics of single-substrate continuous cultures: the role of transport enzymes

A chemostat limited by a single growth-limiting substrate displays a rich spectrum of dynamics. Depending on the flow rate and feed concentration, the chemostat settles into a steady state or executes sustained oscillations. The transients in response to abrupt increases in the flow rate or the feed concentration are also quite complex. For example, if the increase in the flow rate is small, there is no perceptible change in the substrate concentration. If the increase in the flow rate is large, there is a large increase in the substrate concentration lasting several hours or days before the culture adjusts to a new steady state. In the latter case, the substrate concentration and cell density frequently undergo damped oscillations during their approach to the steady state. In this work, we formulate a simple structured model containing the inducible transport enzyme as the key intracellular variable. The model displays the foregoing dynamics under conditions similar to those employed in the experiments. The model suggests that long recovery times (on the order of several hours to several days) can occur because the initial transport enzyme level is too small to cope with the increased substrate supply. The substrate concentration, therefore, increases until the enzyme level is built up to a sufficiently high level by the slow process of enzyme induction. Damped and sustained oscillations can occur because transport enzyme synthesis is autocatalytic, and hence, destabilizing. At low dilution rates, the response of stabilizing processes, such as enzyme dilution and substrate consumption, becomes very slow, leading to damped and sustained oscillations.     

Regulation of ribosome synthesis in Escherichia coli: effects of temperature and dilution rate changes

The effect of temperature on the synthesis of ribosome in Escherichia coli K-12 was investigated. In continuous fermentation, the total and functioning ribosome contents decreased with increasing temperature, while the non-functioning ribosome content remained unchanged. Cells contained higher amounts of functioning ribosome at lower temperatures to compensate for the decrease in translational activity. A transient study was performed to investigate the dynamic response of the cell to changes in the dilution rate. In response to the dilution rate shift-up, the cell mass decreased until the cells produced a sufficient amount of ribosomes to support the new higher growth rate. However, the response to the dilution rate shift-down resulted in an immediate increase in cell mass. This may be due to the fact that the cell already contains enough ribosomes to support a lower growth rate corresponding to the new low dilution rate. Based on the experimental results, a mathematical model was developed to describe the cell growth at transient as well as steady states. The total ribosome content was included as a variable because it affects the growth rate of the cell. (c) 1996 John Wiley & Sons, Inc.     

The rates of macromolecular chain elongation modulate the initiation frequencies for transcription and translation inEscherichia coli

Here we show that most macromolecular biosynthesis reactions in growing bacteria are sub-saturated with substrate. The experiments should in part test predictions from a previously proposed model (Jensen & Pedersen 1990) which proposed a central role for the rates of the RNA and peptide chain elongation reactions in determining the concentration of initiation competent RNA polymerases and ribosomes and thereby the initiation frequencies for these reactions. We have shown that synthesis of ribosomal RNA and the concentration of ppGpp did not exhibit the normal inverse correlation under balanced growth conditions in batch cultures when the RNA chain elongation rate was limited by substrate supply. The RNA chain elongation rate for the polymerase transcribinglacZ mRNA was directly measured and found to be reduced by two-fold under conditions of high ppGpp levels. In the case of translation, we have shown that the peptide elongation rate varied at different types of codons and even among codons read by the same tRNA species. The faster translated codons probably have the highest cognate tRNA concentration and the highest affinity to the tRNA. Thus, the ribosome may operate close to saturation at some codons and be unsaturated at synonymous codons. Therefore, not only translation of the codons for the seven amino acids, whose biosynthesis is regulated by attenuation, but also a substantial fraction of the other translation reactions may be unsaturated. Recently, we have obtained results which indicate that also many ribosome binding sites are unsaturated with their substrate, i.e. with ribosomes. This observation affects the interpretation of many results obtained by use of reporter genes, because the expression from such genes is strongly influenced by the general physiology of the cell.     

Exacting predictions by cybernetic model confirmed experimentally: Steady state multiplicity in the chemostat

We demonstrate strong experimental support for the cybernetic model based on maximizing carbon uptake rate in describing the microorganism's regulatory behavior by verifying exacting predictions of steady state multiplicity in a chemostat. Experiments with a feed mixture of glucose and pyruvate show multiple steady state behavior as predicted by the cybernetic model. When multiplicity occurs at a dilution (growth) rate, it results in hysteretic behavior following switches in dilution rate from above and below. This phenomenon is caused by transient paths leading to different steady states through dynamic maximization of the carbon uptake rate. Thus steady state multiplicity is a manifestation of the nonlinearity arising from cybernetic mechanisms rather than of the nonlinear kinetics. The predicted metabolic multiplicity would extend to intracellular states such as enzyme levels and fluxes to be verified in future experiments. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Different sensitivity of H69 modification enzymes RluD and RlmH to mutations in Escherichia coli 23S rRNA

Nucleoside modifications are introduced into the ribosomal RNA during the assembly of the ribosome. The number and the localization of the modified nucleosides in rRNAs are known for several organisms. In bacteria, rRNA modified nucleosides are synthesized by a set of specific enzymes, the majority of which have been identified in Escherichia coli. Each rRNA modification enzyme recognizes its substrate nucleoside(s) at a specific stage of ribosome assembly. Not much is known about the specificity determinants involved in the substrate recognition of the modification enzymes. In order to shed light on the substrate specificity of RluD and RlmH, the enzymes responsible for the introduction of modifications into the stem-loop 69 (H69), we monitored the formation of H69 pseudouridines (Ψ) and methylated pseudouridine (m3Ψ) in vitro on ribosomes with alterations in 23S rRNA. While the synthesis of Ψs in H69 by RluD is relatively insensitive to the point mutations at neighboring positions, methylation of one of the Ψs by RlmH exhibited a much stronger sensitivity. Apparently, in spite of synthesizing modifications in the same region or even at the same position of rRNA, the two enzymes employ different substrate recognition mechanisms.     

Development,Antibiotic Production,and Ribosome Assembly in Streptomyces venezuelae Are Impacted by RNase J and RNase III Deletion

RNA metabolism is a critical but frequently overlooked control element affecting virtually every cellular process in bacteria. RNA processing and degradation is mediated by a suite of ribonucleases having distinct cleavage and substrate specificity. Here, we probe the role of two ribonucleases (RNase III and RNase J) in the emerging model system Streptomyces venezuelae. We show that each enzyme makes a unique contribution to the growth and development of S. venezuelae and further affects the secondary metabolism and antibiotic production of this bacterium. We demonstrate a connection between the action of these ribonucleases and translation, with both enzymes being required for the formation of functional ribosomes. RNase III mutants in particular fail to properly process 23S rRNA, form fewer 70S ribosomes, and show reduced translational processivity. The loss of either RNase III or RNase J additionally led to the appearance of a new ribosomal species (the 100S ribosome dimer) during exponential growth and dramatically sensitized these mutants to a range of antibiotics.     

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.     

Substrate specificity and properties of the Escherichia coli 16S rRNA methyltransferase, RsmE

The small ribosome subunit of Escherichia coli contains 10 base-methylated sites distributed in important functional regions. At present, seven enzymes responsible for methylation of eight bases are known, but most of them have not been well characterized. One of these enzymes, RsmE, was recently identified and shown to specifically methylate U1498. Here we describe the enzymatic properties and substrate specificity of RsmE. The enzyme forms dimers in solution and is most active in the presence of 10-15 mM Mg(2+) and 100 mM NH(4)Cl at pH 7-9; however, in the presence of spermidine, Mg(2+) is not required for activity. While small ribosome subunits obtained from an RsmE deletion strain can be methylated by purified RsmE, neither 70S ribosomes nor 50S subunits are active. Likewise, 16S rRNA obtained from the mutant strain, synthetic 16S rRNA, and 3' minor domain RNA are all very poor or inactive as substrates. 30S particles partially depleted of proteins by treatment with high concentrations of LiCl or in vitro reconstituted intermediate particles also show little or no methyl acceptor activity. Based on these data, we conclude that RsmE requires a highly structured ribonucleoprotein particle as a substrate for methylation, and that methylation events in the 3' minor domain of 16S rRNA probably occur late during 30S ribosome assembly.     

Visualizing the transfer-messenger RNA as the ribosome resumes translation

Bacterial ribosomes stalled by truncated mRNAs are rescued by transfer-messenger RNA (tmRNA), a dual-function molecule that contains a tRNA-like domain (TLD) and an internal open reading frame (ORF). Occupying the empty A site with its TLD, the tmRNA enters the ribosome with the help of elongation factor Tu and a protein factor called small protein B (SmpB), and switches the translation to its own ORF. In this study, using cryo-electron microscopy, we obtained the first structure of an in vivo-formed complex containing ribosome and the tmRNA at the point where the TLD is accommodated into the ribosomal P site. We show that tmRNA maintains a stable 'arc and fork' structure on the ribosome when its TLD moves to the ribosomal P site and translation resumes on its ORF. Based on the density map, we built an atomic model, which suggests that SmpB interacts with the five nucleotides immediately upstream of the resume codon, thereby determining the correct selection of the reading frame on the ORF of tmRNA.     

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.     

Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. I. Functional tests on rat liver microsomal subfractions

A preparation of rat liver microsomes containing 70% of the total cellular endoplasmic reticulum (ER) membranes was subfractionated by isopycnic density centrifugation. Twelve subfractions of different ribosome content ranging in density from 1.06 to 1.29 were obtained and analyzed with respect to marker enzymes, RNA, and protein content, as well as the capacity of these membranes to bind 80S ribosomes in vitro. After removal of native polysomes from these microsomal subfractions by puromycin in a buffer of high ionic strength their capacity to rebind 80S ribosomes approached levels found in the corresponding native membranes before ribosome stripping. This indicates that in vitro rebinding of ribosomes occurs to the same sites occupied in the cell by membrane-bound polysomes. Microsomes in the microsomal subfractions were also tested for their capacity to effect the translocation of nascent secretory proteins into the microsomal lumen utilizing a rabbit reticulocyte translation system programmed with mRNA coding for the precursor of human placental lactogen. Membranes from microsomes with the higher isopycnic density and a high ribosome content showed the highest translocation activity, whereas membranes derived from smooth microsomes had only a very low translocation activity. These results indicate the membranes of the rough and smooth portions of the endoplasmic reticulum are functionally differentiated so that sites for ribosome binding and the translocation of nascent polypeptides are segregated to the rough domain of the organelle.     

Demyelination induces transport of ribosome-containing vesicles from glia to axons: evidence from animal models and MS patient brains

Glial cells were previously proven capable of trafficking polyribosomes to injured axons. However, the occurrence of such transfer in the general pathological context, such as demyelination-related diseases, needs further evidence. Since this may be a yet unidentified universal contributor to axonal survival, we study putative glia–axonal ribosome transport in response to demyelination in animal models and patients in both peripheral and central nervous system. In the PNS we investigate whether demyelination in a rodent model has the potential to induce ribosome transfer. We also probe the glia–axonal ribosome supply by implantation of transgenic Schwann cells engineered to produce fluorescent ribosomes in the same demyelination model. We furthermore examine the presence of axonal ribosomes in mouse experimental autoimmune encephalomyelitis (EAE), a well-established model for multiple sclerosis (MS), and in human MS autopsy brain material. We provide evidence for increased axonal ribosome content in a pharmacologically demyelinated sciatic nerve, and demonstrate that at least part of these ribosomes originate in the transgenic Schwann cells. In the CNS one of the hallmarks of MS is demyelination, which is associated with severe disruption of oligodendrocyte–axon interaction. Here, we provide evidence that axons from spinal cords of EAE mice, and in the MS human brain contain an elevated amount of axonal ribosomes compared to controls. Our data provide evidence that increased axonal ribosome content in pathological axons is at least partly due to glia-to-axon transfer of ribosomes, and that demyelination in the PNS and in the CNS is one of the triggers capable to initiate this process.     

Protein synthesis inhibitors and catalytic RNA. Effect of puromycin on tRNA precursor processing by the RNA component of Escherichia coli RNase P

RNase P and ribosomes must interact with similar substrate molecules, tRNA precursors in the case of RNase P and aminoacyl-, peptidyl- or free tRNAs in the case of ribosomes. In order to compare the substrate recognition mechanisms between ribosomes and RNase P, protein synthesis inhibitors have been assayed for their effect on the catalytic activity of the RNA component of Escherichia coli RNase P (M1 RNA). Puromycin has an inhibitory effect that could be related to similar substrate recognition mechanisms by rRNA in the ribosome and by M1 RNA in RNase P.     

Error propagation in E. coli protein synthesis

A new approach to the error catastrophe theory, proposed by Leslie Orgel, is presented here. Our model is a development of previous models, but differs in several respects: the overall activity is assumed to be dependent on the error level, the effect of errors in the translating system, giving rise to additional errors in the succeeding generation of products, is explicitly included as a special term in our model, and scavenging enzymes are assumed to break down and eliminate products with a loose structure. Their efficiency is dependent on the error level. The model also takes into account the dilution of incorrect ribosomes and enzymes, and is described by a time-dependence in terms of ribosome/enzyme generations. The model and the contribution to the time development are discussed in the light of experiments on E. coli treated with streptomycin.     

Understanding Biases in Ribosome Profiling Experiments Reveals Signatures of Translation Dynamics in Yeast

Ribosome profiling produces snapshots of the locations of actively translating ribosomes on messenger RNAs. These snapshots can be used to make inferences about translation dynamics. Recent ribosome profiling studies in yeast, however, have reached contradictory conclusions regarding the average translation rate of each codon. Some experiments have used cycloheximide (CHX) to stabilize ribosomes before measuring their positions, and these studies all counterintuitively report a weak negative correlation between the translation rate of a codon and the abundance of its cognate tRNA. In contrast, some experiments performed without CHX report strong positive correlations. To explain this contradiction, we identify unexpected patterns in ribosome density downstream of each type of codon in experiments that use CHX. These patterns are evidence that elongation continues to occur in the presence of CHX but with dramatically altered codon-specific elongation rates. The measured positions of ribosomes in these experiments therefore do not reflect the amounts of time ribosomes spend at each position in vivo. These results suggest that conclusions from experiments in yeast using CHX may need reexamination. In particular, we show that in all such experiments, codons decoded by less abundant tRNAs were in fact being translated more slowly before the addition of CHX disrupted these dynamics.     

Dendritic synchrony and transient dynamics in a coupled oscillator model of the dopaminergic neuron

Transient increases in spontaneous firing rate of mesencephalic dopaminergic neurons have been suggested to act as a reward prediction error signal. A mechanism previously proposed involves subthreshold calcium-dependent oscillations in all parts of the neuron. In that mechanism, the natural frequency of oscillation varies with diameter of cell processes, so there is a wide variation of natural frequencies on the cell, but strong voltage coupling enforces a single frequency of oscillation under resting conditions. In previous work, mathematical analysis of a simpler system of oscillators showed that the chain of oscillators could produce transient dynamics in which the frequency of the coupled system increased temporarily, as seen in a biophysical model of the dopaminergic neuron. The transient dynamics was shown to be consequence of a slow drift along an invariant subset of phase space, with rate of drift given by a Lyapunov function. In this paper, we show that the same mathematical structure exists for the full biophysical model, giving physiological meaning to the slow drift and the Lyapunov function, which is shown to describe differences in intracellular calcium concentration in different parts of the cell. The duration of transients was long, being comparable to the time constant of calcium disposition. These results indicate that brief changes in input to the dopaminergic neuron can produce long lasting firing rate transients whose form is determined by intrinsic cell properties.     

Kinetic and thermodynamic studies of peptidyltransferase in ribosomes from the extreme thermophile Thermus thermophilus

Throughout evolution, emerging organisms survived by adapting existing biochemical processes to new reaction conditions. Simple protein enzymes balanced changes in structural stability with changes that permitted optimal catalysis by adjustments in both entropic and enthalpic contributions to the free energy of activation for the reaction. Study of adaptive mechanisms by large multicomponent enzymes such as the ribosome has been largely unexplored. Here we have determined the kinetic and thermodynamic parameters of peptidyltransferase in ribosomes from the extreme thermophile Thermus thermophilus. Activity of thermophilic enzymes can be assayed over a wide range of temperatures, enabling one to measure accurate catalytic rates and determine enthalpic and entropic contributions to the free energy of activation of the reaction. Differences in the reaction conditions used here and in published studies on mesophilic ribosomes prevent direct comparison, but our data on Thermus ribosomes suggest that these ribosomes have adapted to changing environments using the same strategies as simple protein enzymes, balancing stability and flexibility without loss of catalytic rate. This strategy must be a very ancient process, perhaps first used by primitive ribosomes in the RNA World.     

The role of ribosome recycling factor in dissociation of 70S ribosomes into subunits

Protein synthesis is initiated on ribosomal subunits. However, it is not known how 70S ribosomes are dissociated into small and large subunits. Here we show that 70S ribosomes, as well as the model post-termination complexes, are dissociated into stable subunits by cooperative action of three translation factors: ribosome recycling factor (RRF), elongation factor G (EF-G), and initiation factor 3 (IF3). The subunit dissociation is stable enough to be detected by conventional sucrose density gradient centrifugation (SDGC). GTP, but not nonhydrolyzable GTP analog, is essential in this process. We found that RRF and EF-G alone transiently dissociate 70S ribosomes. However, the transient dissociation cannot be detected by SDGC. IF3 stabilizes the dissociation by binding to the transiently formed 30S subunits, preventing re-association back to 70S ribosomes. The three-factor-dependent stable dissociation of ribosomes into subunits completes the ribosome cycle and the resulting subunits are ready for the next round of translation.