Diauxic shift-dependent relocalization of decapping activators Dhh1 and Pat1 to polysomal complexes

Dhh1 and Pat1 in yeast are mRNA decapping activators/translational repressors thought to play key roles in the transition of mRNAs from translation to degradation. However, little is known about the physical and functional relationships between these proteins and the translation machinery. We describe a previously unknown type of diauxic shift-dependent modulation of the intracellular locations of Dhh1 and Pat1. Like the formation of P bodies, this phenomenon changes the spatial relationship between components involved in translation and mRNA degradation. We report significant spatial separation of Dhh1 and Pat1 from ribosomes in exponentially growing cells. Moreover, biochemical analyses reveal that these proteins are excluded from polysomal complexes in exponentially growing cells, indicating that they may not be associated with active states of the translation machinery. In contrast, under diauxic growth shift conditions, Dhh1 and Pat1 are found to co-localize with polysomal complexes. This work suggests that Dhh1 and Pat1 functions are modulated by a re-localization mechanism that involves eIF4A. Pull-down experiments reveal that the intracellular binding partners of Dhh1 and Pat1 change as cells undergo the diauxic growth shift. This reveals a new dimension to the relationship between translation activity and interactions between mRNA, the translation machinery and decapping activator proteins.     

Effect of different glucose concentrations on proteome of Saccharomyces cerevisiae

We performed a proteomic study to understand how Saccharomyces cerevisiae adapts its metabolism during the exponential growth on three different concentrations of glucose; this information will be necessary to understand yeast carbon metabolism in different environments. We induced a natural diauxic shift by growing yeast cells in glucose restriction thus having a fast and complete glucose exhaustion. We noticed differential expressions of groups of proteins. Cells in high glucose have a decreased growth rate during the initial phase of fermentation; in glucose restriction and in high glucose we found an over-expression of a protein (Peroxiredoxin) involved in protection against oxidative stress insult. The information obtained in our study validates the application of a proteomic approach for the identification of the molecular bases of environmental variations such as fermentation in high glucose and during a naturally induced diauxic shift.     

Contributions of XylR,CcpA and cre to diauxic growth of Bacillus megaterium and to xylose isomerase expression in the presence of glucose and xylose

Bacillus megaterium shows diauxic growth in minimal medium containing glucose and xylose. We have examined the influence of three elements that regulate xyl operon expression on diauxic growth and expression of a xylA-lacZ fusion. xylA is 13-fold repressed during growth on glucose. Induction occurs at the onset of the lag phase after glucose is consumed. Inactivation of xylR yields a two-fold increase in expression of xylA on glucose. Deletion of the catabolite responsive element (cre) has a more pronounced effect, reducing glucose repression from 13-fold in the wild type to about 2.5-fold. When xylR and cre are inactivated together a residual two-fold repression of xylA is found. Inactivation of xylR affects diauxic growth by shortening the lag phase from 70 to 40?min. In-frame deletion of ccpA results in the loss of diauxic growth, an increase in doubling time and simultaneous use of both sugars. In contrast, a strain with an inactivated cre site in xylA exhibits diauxic growth without an apparent lag phase on glucose and xylose, whereas fructose and xylose are consumed simultaneously.     

Changes in gene expression in the Ras/adenylate cyclase system of Saccharomyces cerevisiae: correlation with cAMP levels and growth arrest.

Levels of cyclic 3',5'-cyclic monophosphate (cAMP) play an important role in the decision to enter the mitotic cycle in the yeast, Saccharomyces cerevisiae. In addition to growth arrest at stationary phase, S. cerevisiae transiently arrest growth as they shift from fermentative to oxidative metabolism (the diauxic shift). Experiments examining the role of cAMP in growth arrest at the diauxic shift show the following: 1) yeast lower cAMP levels as they exhaust their glucose supply and shift to oxidative metabolism of ethanol, 2) a reduction in cAMP is essential for traversing the diauxic shift, 3) the decrease in adenylate cyclase activity is associated with a decrease in the expression of CYR1 and CDC25, two positive regulators of cAMP levels and an increase in the expression of IRA1 and IRA2, two negative regulators of intracellular cAMP, 4) mutants carrying disruptions in IRA1 and IRA2 were unable to arrest cell division at the diauxic shift and were unable to progress into the oxidative phase of growth. These results indicate that changes cAMP levels are important in regulation of growth arrest at the diauxic shift and that changes in gene expression plays a role in the regulation of the Ras/adenylate cyclase system.     

Single-Cell Dynamics Reveals Sustained Growth during Diauxic Shifts

Stochasticity in gene regulation has been characterized extensively, but how it affects cellular growth and fitness is less clear. We study the growth of E. coli cells as they shift from glucose to lactose metabolism, which is characterized by an obligatory growth arrest in bulk experiments that is termed the lag phase. Here, we follow the growth dynamics of individual cells at minute-resolution using a single-cell assay in a microfluidic device during this shift, while also monitoring lac expression. Mirroring the bulk results, the majority of cells displays a growth arrest upon glucose exhaustion, and resume when triggered by stochastic lac expression events. However, a significant fraction of cells maintains a high rate of elongation and displays no detectable growth lag during the shift. This ability to suppress the growth lag should provide important selective advantages when nutrients are scarce. Trajectories of individual cells display a highly non-linear relation between lac expression and growth, with only a fraction of fully induced levels being sufficient for achieving near maximal growth. A stochastic molecular model together with measured dependencies between nutrient concentration, lac expression level, and growth accurately reproduces the observed switching distributions. The results show that a growth arrest is not obligatory in the classic diauxic shift, and underscore that regulatory stochasticity ought to be considered in terms of its impact on growth and survival.     

Single Bacteria Movement Tracking by Online Microscopy – A Proof of Concept Study

In this technical report we demonstrate a low-cost online unit allowing movement tracking of flagellated bacteria on a single-cell level during fermentation processes. The system’s ability to distinguish different metabolic states (viability) of bacteria by movement velocity was investigated. A flow-through cuvette with automatically adjustable layer thickness was developed. The cuvette can be used with most commercially available laboratory microscopes equipped with 40× amplification and a digital camera. In addition, an automated sample preparation unit and a software module was developed measuring size, moved distance, and speed of bacteria. In a proof of principle study the movement velocities of Bacillus amyloliquefaciens FZB42 during three batch fermentation processes were investigated. In this process the bacteria went through different metabolic states, vegetative growth, diauxic shift, vegetative growth after diauxic shift, and sporulation. It was shown that the movement velocities during the different metabolic states significantly differ from each other. Therefore, the described setup has the potential to be used as a bacteria viability monitoring tool. In contrast to some other techniques, such as electro-optical techniques, this method can even be used in turbid production media.     

Diauxic lags explain unexpected coexistence in multi‐resource environments

How the coexistence of species is affected by the presence of multiple resources is a major question in microbial ecology. We experimentally demonstrate that differences in diauxic lags, which occur as species deplete their own environments and adapt their metabolisms, allow slow‐growing microbes to stably coexist with faster‐growing species in multi‐resource environments despite being excluded in single‐resource environments. In our focal example, an Acinetobacter species (Aci2) competitively excludes Pseudomonas aurantiaca (Pa) on alanine and on glutamate. However, they coexist on the combination of both resources. Experiments reveal that Aci2 grows faster but Pa has shorter diauxic lags. We establish a tradeoff between Aci2’s fast growth and Pa’s short lags as their mechanism for coexistence. We model this tradeoff to accurately predict how environmental changes affect community composition. We extend our work by surveying a large set of competitions and observe coexistence nearly four times as frequently when the slow‐grower is the fast‐switcher. Our work illustrates a simple mechanism, based entirely on supplied‐resource growth dynamics, for the emergence of multi‐resource coexistence.     

Suppression of rpsL phenotypes by tuf mutations reveals a unique relationship between translation elongation and growth rate

We have found a simple relationship between bacterial growth rate and the translation elongation rate. Thus, for a set of defined ribosomal protein S12 mutations which reduce the efficiency of the ternary complex ribosome interaction (and restrict the frequency of translational errors) there is a linear relationship between growth rate and translation elongation rate. When these mutants are combined with defined EF-Tu mutants (which increase the probability of translational errors) both the elongation rate and growth rate reductions are reversed. The reductions and reversals are described by a unique linear relationship. We interpret this to mean that these two types of mutation exert opposing effects on the same molecular interaction. We suggest that this interaction is in the initial selection of the aminoacyl-tRNA on the ribosome. The slope of the relationship between translation elongation rate and growth rate, defined in per cent of the wild-type rates, is close to 1. Interestingly, the reversal of the elongation and growth phenotypes is Incomplete, suggesting that the ribosomal mutants have an additional defect which is not compensated for by the ternary complex interaction. Our results show that the efficiency of the ternary complex ribosome interaction limits the translation elongation rate, which in turn correlates with changes in exponential growth rate.     

Preferential uptake of d-α-aminoadipate from a racemic mixture by an Alcaligenes denitrificans

During growth on racemic α-aminoadipate as a sole source of carbon and nitrogen an Alcaligenes denitrificans displayed a two-phase (diauxic) growth not shown by a Pseudomonas putida. Oxidation studies in the presence of chloramphenicol showed that in both growth phases whole cells of A. denitrificans had the ability to oxidize either isomer of α-aminoadipate. These data negated as the physiological basis for diauxic growth a proposal that one isomer was repressing the synthesis of enzymes which transport or degrade the other enantiomer. Growth studies with various ratios of the d- and l-isomers of α-aminoadipate, differentiating one isomer from the other by radioactivity, indicated that diauxic growth resulted from a preferential uptake of d-α-aminoadipate over the l-isomer.     

Metabolic alterations in yeast lacking copper-zinc superoxide dismutase

Yeast lacking copper-zinc superoxide dismutase (sod1?) have a number of oxygen-dependent defects, including auxotrophies for lysine and methionine and sensitivity to oxygen. Here we report additional defects in metabolic regulation. Under standard growth conditions with glucose as the carbon source, yeast undergo glucose repression in which mitochondrial respiration is deemphasized, energy is mainly derived from glycolysis, and ethanol is produced. When glucose is depleted, the diauxic shift is activated, in which mitochondrial respiration is reemphasized and stress resistance increases. We find that both of these programs are adversely affected by the lack of Sod1p. Key events in the diauxic shift do not occur and sod1? cells do not utilize ethanol and stop growing. The ability to shift to growth on ethanol is gradually lost as time in culture increases. In early stages of culture, sod1? cells consume more oxygen and have more mitochondrial mass than wild-type cells, indicating that glucose repression is not fully activated. These changes are at least partially dependent on the activity of the Hap2,3,4,5 complex, as indicated by CYC1-lacZ reporter assays. These changes may indicate a role for superoxide in metabolic signaling and regulation and/or a role for glucose derepression in defense against oxidative stress.     

A comprehensive analysis of translational missense errors in the yeast Saccharomyces cerevisiae

The process of protein synthesis must be sufficiently rapid and sufficiently accurate to support continued cellular growth. Failure in speed or accuracy can have dire consequences, including disease in humans. Most estimates of the accuracy come from studies of bacterial systems, principally Escherichia coli, and have involved incomplete analysis of possible errors. We recently used a highly quantitative system to measure the frequency of all types of misreading errors by a single tRNA in E. coli. That study found a wide variation in error frequencies among codons; a major factor causing that variation is competition between the correct (cognate) and incorrect (near-cognate) aminoacyl-tRNAs for the mutant codon. Here we extend that analysis to measure the frequency of missense errors by two tRNAs in a eukaryote, the yeast Saccharomyces cerevisiae. The data show that in yeast errors vary by codon from a low of 4 × 10⁻⁵ to a high of 6.9 × 10⁻⁴ per codon and that error frequency is in general about threefold lower than in E. coli, which may suggest that yeast has additional mechanisms that reduce missense errors. Error rate again is strongly influenced by tRNA competition. Surprisingly, missense errors involving wobble position mispairing were much less frequent in S. cerevisiae than in E. coli. Furthermore, the error-inducing aminoglycoside antibiotic, paromomycin, which stimulates errors on all error-prone codons in E. coli, has a more codon-specific effect in yeast.