David C. J. Gardner

Growth control of the eukaryote cell: a systems biology study in yeast

BACKGROUND Cell growth underlies many key cellular and developmental processes, yet a limited number of studies have been carried out on cell-growth regulation. Comprehensive studies at the transcriptional, proteomic and metabolic levels under defined controlled conditions are currently lacking. RESULTS Metabolic control analysis is being exploited in a systems biology study of the eukaryotic cell. Using chemostat culture, we have measured the impact of changes in flux (growth rate) on the transcriptome, proteome, endometabolome and exometabolome of the yeast Saccharomyces cerevisiae. Each functional genomic level shows clear growth-rate-associated trends and discriminates between carbon-sufficient and carbon-limited conditions. Genes consistently and significantly upregulated with increasing growth rate are frequently essential and encode evolutionarily conserved proteins of known function that participate in many protein-protein interactions. In contrast, more unknown, and fewer essential, genes are downregulated with increasing growth rate; their protein products rarely interact with one another. A large proportion of yeast genes under positive growth-rate control share orthologs with other eukaryotes, including humans. Significantly, transcription of genes encoding components of the TOR complex (a major controller of eukaryotic cell growth) is not subject to growth-rate regulation. Moreover, integrative studies reveal the extent and importance of post-transcriptional control, patterns of control of metabolic fluxes at the level of enzyme synthesis, and the relevance of specific enzymatic reactions in the control of metabolic fluxes during cell growth. CONCLUSION This work constitutes a first comprehensive systems biology study on growth-rate control in the eukaryotic cell. The results have direct implications for advanced studies on cell growth, in vivo regulation of metabolic fluxes for comprehensive metabolic engineering, and for the design of genome-scale systems biology models of the eukaryotic cell.

Dispersed growth of Streptomyces in liquid culture

SummaryThe study of the physiology of the filamentous bacterium Streptomyces is inhibited by its formation of mycelial pellets in liquid cultures. It is demonstrated that dispersed growth may be achieved by the addition of polymers to the culture medium. Uncharged polymers, such as polyethylene glycol, are relatively ineffective but polyanions such as agar, Carbopol and Junlon produce dispersed cultures when included in a defined growth medium at low concentrations. Junlon-containing media enable optical density measurements to be used to follow batch growth of Streptomyces. Improvements in both biomass yield and product yield of the pigmented antibiotic actinorhodin were found to result from the incorporation of Junlon into minimal medium.

Suitability of replacement markers for functional analysis studies in Saccharomyces cerevisiae

The complete yeast sequence contains a large proportion of genes whose biological function is completely unknown. One approach to elucidating the function of these novel genes is by quantitative methods that exploit the concepts of metabolic control analysis. An important first step in such an analysis is to determine the effects of deleting individual genes on the growth rate (or fitness) of Saccharomyces cerevisiae. Since the specific growth‐rate effects of most genes are likely to be small, they are most readily determined by competition against a standard strain in chemostat cultures where the true steady state demanded by metabolic control analysis may be achieved. We have constructed two different standard strains in which the HO gene is replaced by either HIS3 or kanMX. We demonstrate that HO is a selectively neutral site for gene replacement. However, there is a significant marker effect associated with HIS3 which, moreover, is dependent on the physiological conditions used for the competition experiments. In contrast, the kanMX marker exhibited only a small effect on specific growth rate (≤±4%). These data suggest that nutritional markers should not be used to generate deletion mutants for the quantitative analysis of gene function in yeast but that kanMX replacements may be used, with confidence, for such studies.

PIGMENTED ANTIBIOTIC PRODUCTION BY STREPTOMYCES COELICOLOR A3(2) : KINETICS AND THE INFLUENCE OF NUTRIENTS

SUMMARY: The production of the pigments actinorhodin and undecylprodigiosin by Streptomyces coelicolor A3(2) was examined in a chemically defined medium which permits dispersed growth of the organism. The physiological controls on the production of the two pigments were markedly disparate. Actinorhodin production occurred mainly in the stationary phase of batch cultures grown with glucose and sodium nitrate as the principal carbon and nitrogen sources. In the same batch cultures, undecylprodigiosin accumulated during the exponential growth phase. The production of both pigments was sensitive to the levels of ammonium and phosphate in the medium. Actinorhodin production was exquisitely sensitive to ammonium concentration, and was completely inhibited by as little as 1 mM-ammonium chloride, whereas more than 50 mM-ammonium chloride was required to prevent undecylprodigiosin production. A similar, but less extreme effect was seen with phosphate: actinorhodin production was completely inhibited by 24 mM-phosphate, whereas undecylprodigiosin was still formed at this high phosphate concentration. The effects of ammonium inhibition of pigmented antibiotic production were relieved by reducing the concentration of phosphate in the medium, but changing the ammonium concentration had no effect on phosphate inhibition. Thus the regulation of pigment production by these two nutrients is interrelated, with phosphate control being epistatic to that of ammonium. The results implicate a phosphorylated intermediate as a major regulator of secondary metabolite synthesis by S. coelicolor.

Stability of a cloned gene in yeast grown in chemostat culture.

SummaryA study has been made of the stability of LEU2, a cloned chromosomal gene of Saccharomyces cerevisiae, when reintroduced into yeast on a number of plasmid vectors which permit a chromosomal or episomal location for the gene in either high or low copy number. Glucose-limited continuous culture was employed to ensure that there was no selection for the inserted gene. Both the rate of segregation of plasmid minus cells and the effect of the plasmid on host growth rate were found to determine plasmid stability which, in many cases, could be predicted by simple mathematical models. The presence or absence of the endogenous 2 μ plasmid of yeast was found to have an important influence on the stability of 2 μ-based vectors. This led to the discovery that, for the host strain used, the presence of 2 μ sequences represented a selective advantage for the cells.

The yeast 2*-plasmid: strategies for the survival of a selfish DNA

SummaryThe designation of the yeast 2 μ circle as a “selfish” DNA molecule has been confirmed by demonstrating that the plasmid is lost with exponential kinetics from haploid yeast populations grown in continuous culture. We show that plasmid-free yeast cells have a growth rate advantage of some 1.5%–3% over their plasmid-containing counterparts. This finding makes the ubiquity of this selfish DNA in yeast strains puzzling. Two other factors probably account for its survival. First, the rate of plasmid loss was reduced by allowing haploid populations to enter stationary phase periodically. Second, it was not possible to isolate a plasmid-free segregant from a diploid yeast strain. Competition experiments demonstrated that stability in a diploid is conferred at the level of segregation and that plasmid-free diploid cells are at a selective advantage compared with their plasmid-containing counterparts. Yeast cells in nature are usually homothallic and must frequently pass through both diploid and stationary phases. The 2 μ plasmid appears to have evolved a survival strategy which exploits these two features of its hosts life cycle.

Quantitative analysis of yeast gene function using competition experiments in continuous culture

One possible route to the evaluation of gene function is a quantitative approach based on the concepts of metabolic control analysis (MCA). An important first step in such an analysis is to determine the effect of deleting individual genes on the growth rate (or fitness) of S. cerevisiae. Since the specific growth‐rate effects of most genes are likely to be small, we employed competition experiments in chemostat culture to measure the proportion of deletion mutants relative to that of a standard strain by using a quantitative PCR method. In this paper, we show that both densitometry and GeneScan™ analysis can be used with similar accuracy and reproducibility to determine the proportions of (at least) two strains simultaneously, in the range 10–90% of the total cell population. Furthermore, we report on a model competition experiment between two diploid nuclear petite mutants, homozygous for deletions in the cox5a or pet191 genes, and the standard strain (ho::kanMX4/ho::kanMX4) in chemostat cultures under six different physiological conditions. The results indicate that competition experiments in continuous culture are a suitable method to distinguish quantitatively between deletion mutants that qualitatively exhibit the same phenotype.

Disruption of seven hypothetical aryl alcohol dehydrogenase genes from Saccharomyces cerevisiae and construction of a multiple knock-out strain

By in silicio analysis, we have discovered that there are seven open reading frames (ORFs) in Saccharomyces cerevisiae whose protein products show a high degree of amino acid sequence similarity to the aryl alcohol dehydrogenase (AAD) of the lignin‐degrading fungus Phanerochaete chrysosporium. Yeast cultures grown to stationary phase display a significant aryl alcohol dehydrogenase activity by degrading aromatic aldehydes to the corresponding alcohols. To study the biochemical and the biological role of each of the AAD genes, a series of mutant strains carrying deletion of one or more of the AAD‐coding sequences was constructed by PCR‐mediated gene replacement, using the readily selectable marker kanMX. The correct targeting of the PCR‐generated disruption cassette into the genomic locus was verified by analytical PCR and by pulse‐field gel electrophoresis (PFGE) followed by Southern blot analysis. Double, triple and quadruple mutant strains were obtained by classical genetic methods, while the construction of the quintuple, sextuple and septuple mutants was achieved by using the marker URA3 from Kluyveromyces lactis, HIS3 from Schizosaccharomyces pombe and TRP1 from S. cerevisiae. None of the knock‐out strains revealed any mutant phenotype when tested for the degradation of aromatic aldehydes using both spectrophotometry and high performance liquid chromatography (HPLC). Specific tests for changes in the ergosterol and phospholipids profiles did not reveal any mutant phenotype and mating and sporulation efficiencies were not affected in the septuple deletant. Compared to the wild‐type strain, the septuple deletant showed an increased resistance to the anisaldehyde, but there is a possibility that the nutritional markers used for gene replacement are causing this effect. Copyright

Transcript analysis of 1003 novel yeast genes using high-throughput northern hybridizations

The expression of 1008 open reading frames (ORFs) from the yeast Saccharomyces cerevisiae has been examined under eight different physiological conditions, using classical northern analysis. These northern data have been compared with publicly available data from a microarray analysis of the diauxic transition in S.cerevisiae. The results demonstrate the importance of comparing biologically equivalent situations and of the standardization of data normalization procedures. We have also used our northern data to identify co‐regulated gene clusters and define the putative target sites of transcriptional activators responsible for their control. Clusters containing genes of known function identify target sites of known activators. In contrast, clusters comprised solely of genes of unknown function usually define novel putative target sites. Finally, we have examined possible global controls on gene expression. It was discovered that ORFs that are highly expressed following a nutritional upshift tend to employ favoured codons, whereas those overexpressed in starvation conditions do not. These results are interpreted in terms of a model in which competition between mRNA molecules for translational capacity selects for codons translated by abundant tRNAs.

Suppression of sorbitol dependence in a strain bearing a mutation in the SRB1/PSA1/VIG9 gene encoding GDP-mannose pyrophosphorylase by PDE2 overexpression suggests a role for the Ras/cAMP signal-transduction pathway in the control of yeast cell-wall biogenesis

Complementation studies and allele replacement in Saccharomyces cerevisiae revealed that PSA1/VIG9, an essential gene that encodes GDP-mannose pyrophosphorylase, is the wild-type SRB1 gene. Cloning and sequencing of the srb1-1 allele showed that it determines a single amino acid change from glycine to aspartic acid at residue 276 (srb1(D276)). Genetic evidence is presented showing that at least one further mutation is required for the sorbitol dependence of srb1(D276). A previously reported complementing gene, which this study has now identified as PDE2, is a multi-copy suppressor of sorbitol dependence and is not, as was previously suggested, the SRB1 gene. srb and pde2 mutants share a number of phenotypes, including lysis upon hypotonic shock and enhanced transformability. These data are consistent with the idea that the Ras/cAMP pathway might modulate cell-wall construction.