Measuring effect of mutations & conditions on microbial respiratory rates.

Cellular respiration is central to a wide range of cellular processes. In microorganisms, the effect of a mutation or an environmental condition on the rate of respiration is usually determined by measuring oxygen consumption in the media. We describe this method and discuss caveats and controls for the method.

Investigating cell autonomy in microorganisms.

Cell-cell signaling in microorganisms is still poorly characterized. In this Methods paper, we describe a genetic procedure for detecting cell-nonautonomous genetic effects, and in particular cell-cell signaling, termed the chimeric colony assay (CCA). The CCA measures the effect of a gene on a biological response in a neighboring cell. This assay can measure cell autonomy for range of biological activities including transcript or protein accumulation, subcellular localization, and cell differentiation. To date, the CCA has been used exclusively to investigate colony patterning in the budding yeast Saccharomyces cerevisiae. To demonstrate the wider potential of the assay, we applied this assay to two other systems: the effect of Grr1 on glucose repression of GAL1 transcription in yeast and the effect of rpsL on stop-codon translational readthrough in Escherichia coli. We also describe variations of the standard CCA that address specific aspects of cell-cell signaling, and we delineate essential controls for this assay. Finally, we discuss complementary approaches to the CCA. Taken together, this Methods paper demonstrates how genetic assays can reveal and explore the roles of cell-cell signaling in microbial processes.

How Boundaries Form: Linked Nonautonomous Feedback Loops Regulate Pattern Formation in Yeast Colonies.

Under conditions in which budding yeast form colonies and then undergo meiosis/sporulation, the resulting colonies are organized such that a sharply defined layer of meiotic cells overlays a layer of unsporulated cells termed "feeder cells." This differentiation pattern requires activation of both the Rlm1/cell-wall integrity pathway and the Rim101/alkaline-response pathway. In the current study, we analyzed the connection between these two signaling pathways in regulating colony development by determining expression patterns and cell-autonomy relationships. We present evidence that two parallel cell-nonautonomous positive-feedback loops are active in colony patterning, an Rlm1-Slt2 loop active in feeder cells and an Rim101-Ime1 loop active in meiotic cells. The Rlm1-Slt2 loop is expressed first and subsequently activates the Rim101-Ime1 loop through a cell-nonautonomous mechanism. Once activated, each feedback loop activates the cell fate specific to its colony region. At the same time, cell-autonomous mechanisms inhibit ectopic fates within these regions. In addition, once the second loop is active, it represses the first loop through a cell-nonautonomous mechanism. Linked cell-nonautonomous positive-feedback loops, by amplifying small differences in microenvironments, may be a general mechanism for pattern formation in yeast and other organisms.

Shrinking Daughters: Rlm1-Dependent G1/S Checkpoint Maintains Saccharomyces cerevisiae Daughter Cell Size and Viability.

The Rlm1 transcription factor is a target of the cell wall integrity pathway. We report that an rlm1Δ mutant grown on a nonfermentable carbon source at low osmolarity forms cell groups in which a mother cell is surrounded by smaller "satellite-daughter" cells. Mother cells in these groups progressed through repeated rounds of cell division with normal rates of bud growth and genetic stability; however, these cells underwent precocious START relative to wild-type mothers. Thus, once activated, Rlm1 delays the transition from G1 to S, a mechanism we term the cell wall/START (CW/START) checkpoint. The rlm1Δ satellite-cell phenotype is suppressed by deletion of either SLT2, which encodes the kinase that activates Rlm1, or SWI4, which is also activated by Slt2; suggesting that Slt2 can have opposing roles in regulating the START transition. Consistent with an Rlm1-dependent CW/START checkpoint, rlm1Δ satellite daughters were unable to grow or divide further even after transfer to rich medium, but UV irradiation in G1 could partially rescue rlm1Δ satellite daughters in the next division. Indeed, after cytokinesis, these satellite daughters shrank rapidly, displayed amorphous actin staining, and became more permeable. As a working hypothesis, we propose that duplication of an "actin-organizing center" in late G1 may be required both to progress through START and to reestablish the actin cytoskeleton in daughter cells.

Similar environments but diverse fates: Responses of budding yeast to nutrient deprivation.

Diploid budding yeast (Saccharomyces cerevisiae) can adopt one of several alternative differentiation fates in response to nutrient limitation, and each of these fates provides distinct biological functions. When different strain backgrounds are taken into account, these various fates occur in response to similar environmental cues, are regulated by the same signal transduction pathways, and share many of the same master regulators. I propose that the relationships between fate choice, environmental cues and signaling pathways are not Boolean, but involve graded levels of signals, pathway activation and master-regulator activity. In the absence of large differences between environmental cues, small differences in the concentration of cues may be reinforced by cell-to-cell signals. These signals are particularly essential for fate determination within communities, such as colonies and biofilms, where fate choice varies dramatically from one region of the community to another. The lack of Boolean relationships between cues, signaling pathways, master regulators and cell fates may allow yeast communities to respond appropriately to the wide range of environments they encounter in nature.

Phenotypic plasticity within yeast colonies: differential partitioning of cell fates.

Across many phyla, a common aspect of multicellularity is the organization of different cell types into spatial patterns. In the budding yeast Saccharomyces cerevisiae, after diploid colonies have completed growth, they differentiate to form alternating layers of sporulating cells and feeder cells. In the current study, we found that as yeast colonies developed, the feeder cell layer was initially separated from the sporulating cell layer. Furthermore, the spatial pattern of sporulation in colonies depended on the colony's nutrient environment; in two environments in which overall colony sporulation efficiency was very similar, the pattern of feeder and sporulating cells within the colony was very different. As noted previously, under moderately suboptimal conditions for sporulation-low acetate concentration or high temperature-the number of feeder cells increases as does the dependence of sporulation on the feeder-cell transcription factor, Rlm1. Here we report that even under a condition that is completely blocked sporulation, the number of feeder cells still increased. These results suggest broader implications to our recently proposed "Differential Partitioning provides Environmental Buffering" or DPEB hypothesis.

Cell Differentiation and Spatial Organization in Yeast Colonies: Role of Cell-Wall Integrity Pathway.

Many microbial communities contain organized patterns of cell types, yet relatively little is known about the mechanism or function of this organization. In colonies of the budding yeast Saccharomyces cerevisiae, sporulation occurs in a highly organized pattern, with a top layer of sporulating cells sharply separated from an underlying layer of nonsporulating cells. A mutant screen identified the Mpk1 and Bck1 kinases of the cell-wall integrity (CWI) pathway as specifically required for sporulation in colonies. The CWI pathway was induced as colonies matured, and a target of this pathway, the Rlm1 transcription factor, was activated specifically in the nonsporulating cell layer, here termed feeder cells. Rlm1 stimulates permeabilization of feeder cells and promotes sporulation in an overlying cell layer through a cell-nonautonomous mechanism. The relative fraction of the colony apportioned to feeder cells depends on nutrient environment, potentially buffering sexual reproduction against suboptimal environments.

GAL1-SceI directed site-specific genomic (gsSSG) mutagenesis: a method for precisely targeting point mutations in S. cerevisiae.

BACKGROUND: Precise targeted mutations are defined as targeted mutations that do not require the retention of other genetic changes, such as marker genes, near the mutation site. In the yeast, S. cerevisiae, there are several methods for introducing precise targeted mutations, all of which depend on inserting both a counter-selectable marker and DNA bearing the mutation. For example, the marker can first be inserted, and then replaced with either a long oligonucleotide carrying the mutation (delitto perfetto) or a PCR fragment synthesized with one primer containing the mutation (SSG mutagenesis). RESULTS: A hybrid method for targeting precise mutation into the genomes uses PCR fragments as in SSG mutagenesis together with a CORE cassette devised for delitto perfetto that contains the homing endonuclease SceI. This method, termed gsSSG mutagenesis, is much more efficient than standard SSG mutagenesis, allowing replacements to be identified without extensive screening of isolates. In gsSSG, recombination between the PCR fragment and the genome occurs equally efficiently regardless of the size of the fragment or the distance between the fragment end and the site of marker insertion. In contrast, the efficiency of incorporating targeted mutations by this method increases as the distance between the mutation and the marker insertion site decreases. CONCLUSION: gsSSG is an efficient way of introducing precise mutations into the genome of S. cerevisiae. The frequency of incorporating the targeted mutation remains efficient at least as far as 460 bp from the insertion site meaning that a single insertion can be used to create many different mutants. The overall efficiency of gsSSG can be estimated based on the distance between the mutation and the marker insertion, and this efficiency can be maximized by limiting the number of untargeted mutations. Thus, a single insertion of marker genes plus homing endonuclease cassette can be used to efficiently introduce precise point mutations through a region of > 900 bp.

Yeast colony embedding method.

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood. One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 µ) useful for light microscopy and thin sections (0.1 µ) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM. The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

Cell signals, cell contacts, and the organization of yeast communities.

Even relatively simple species have evolved mechanisms to organize individual organisms into communities, such that the fitness of the group is greater than the fitness of isolated individuals. Within the fungal kingdom, the ability of many yeast species to organize into communities is crucial for their growth and survival, and this property has important impacts both on the economy and on human health. Over the last few years, studies of Saccharomyces cerevisiae have revealed several fundamental properties of yeast communities. First, strain-to-strain variation in the structures of these groups is attributable in part to variability in the expression and functions of adhesin proteins. Second, the extracellular matrix surrounding these communities can protect them from environmental stress and may also be important in cell signaling. Finally, diffusible signals between cells contribute to community organization so that different regions of a community express different genes and adopt different cell fates. These findings provide an arena in which to view fundamental mechanisms by which contacts and signals between individual organisms allow them to assemble into functional communities.

Flo11p adhesin required for meiotic differentiation in Saccharomyces cerevisiae minicolonies grown on plastic surfaces.

Saccharomyces cerevisiae grown on plastic surfaces formed organized structures, termed minicolonies, that consisted of a core of round (yeast-like) cells surrounded by chains of filamentous cells (pseudohyphae). Minicolonies had a much higher affinity for plastic than unstructured yeast communities growing on the same surface. Pseudohyphae at the surface of these colonies developed further into chains of asci. These structures suggest that pseudohyphal differentiation and sporulation are sequential processes in minicolonies. Consistent with this idea, minicolonies grown under conditions that stimulated pseudohyphal differentiation contained higher frequencies of asci. Furthermore, a flo11Δ mutant, which fails to form pseudohyphae, yielded normal sporulation in cultures, but was defective for minicolony sporulation. When minicolonies were dispersed in water and cells were then allowed to settle on the plastic surface, these cells sporulated very efficiently. Taken together, our results suggest that sporulation in minicolonies is stimulated by pseudohyphal differentiation because these pseudohyphae are dispersed from the core of the colony.

Sporulation patterning and invasive growth in wild and domesticated yeast colonies.

Different cell types can form patterns within fungal communities; for example, colonies of Saccharomyces cerevisiae form two sharply defined layers of sporulating cells separated by an intervening layer of unsporulated cells. Because colony sporulation patterns have only been investigated in a single laboratory strain background (W303), in this report we examined these patterns in other strain backgrounds. Two other laboratory strain backgrounds (SK1 and Sigma1278b) that differ from W303 with respect to colony morphology, invasive growth, and sporulation efficiency nevertheless displayed the same colony sporulation pattern as W303. This pattern was also observed in colonies of wild isolates of S. cerevisiae and Saccharomyces paradoxus. The wild yeast colonies sporulated on a much wider range of carbon sources than did the lab yeast and displayed a similar layered sporulation pattern when grown on either acetate or glucose medium and on either rich or synthetic medium. SK1, Sigma1278b and wild yeast colonies invaded the agar surface. The region of invasion varied between strains with respect to the organization and appearance of cells, but this invasion was always accompanied by sporulation. Thus, sporulation patterns are a general property of S. cerevisiae, and sporulation in colonies can be coordinated with invasive growth.

The Rim101p/PacC pathway and alkaline pH regulate pattern formation in yeast colonies.

Multicellular organisms utilize cell-to-cell signals to build patterns of cell types within embryos, but the ability of fungi to form organized communities has been largely unexplored. Here we report that colonies of the yeast Saccharomyces cerevisiae formed sharply divided layers of sporulating and nonsporulating cells. Sporulation initiated in the colony's interior, and this region expanded upward as the colony matured. Two key activators of sporulation, IME1 and IME2, were initially transcribed in overlapping regions of the colony, and this overlap corresponded to the initial sporulation region. The development of colony sporulation patterns depended on cell-to-cell signals, as demonstrated by chimeric colonies, which contain a mixture of two strains. One such signal is alkaline pH, mediated through the Rim101p/PacC pathway. Meiotic-arrest mutants that increased alkali production stimulated expression of an early meiotic gene in neighboring cells, whereas a mutant that decreased alkali production (cit1Delta) decreased this expression. Addition of alkali to colonies accelerated the expansion of the interior region of sporulation, whereas inactivation of the Rim101p pathway inhibited this expansion. Thus, the Rim101 pathway mediates colony patterning by responding to cell-to-cell pH signals. Cell-to-cell signals coupled with nutrient gradients may allow efficient spore formation and spore dispersal in natural environments.

Glucose induction pathway regulates meiosis in Saccharomyces cerevisiae in part by controlling turnover of Ime2p meiotic kinase.

Several components of the glucose induction pathway, namely the Snf3p glucose sensor and the Rgt1p and Mth1p transcription factors, were shown to be involved in inhibition of sporulation by glucose. The glucose sensors had only a minor role in regulating transcript levels of the two key regulators of meiotic initiation, the Ime1p transcription factor and the Ime2p kinase, but a major role in regulating Ime2p stability. Interestingly, Rgt1p was involved in glucose inhibition of spore formation but not inhibition of Ime2p stability. Thus, the glucose induction pathway may regulate meiosis through both RGT1-dependent and RGT1-independent pathways.

Two-step method for constructing unmarked insertions, deletions and allele substitutions in the yeast genome.

We describe three extensions of the method of site-specific genomic (SSG) mutagenesis. These three extensions of SSG mutagenesis were used to generate precise insertion, deletion, and allele substitution mutations in the genome of the budding yeast, Saccharomyces cerevisiae. These mutations are termed precise because no attached sequences (e.g., marker genes or recombination sites) are retained once the method is complete. Because the method is PCR-based, neither DNA cloning nor synthesis of long oligonucleotides is required. We demonstrated the efficacy of these methods by deleting an ORF, inserting the tandem affinity purification (TAP) tag, and replacing a wild-type allele with a mutant allele.

Glucose inhibits meiotic DNA replication through SCFGrr1p-dependent destruction of Ime2p kinase.

In the budding yeast Saccharomyces cerevisiae, the cell division cycle and sporulation are mutually exclusive cell fates; glucose, which stimulates the cell division cycle, is a potent inhibitor of sporulation. Addition of moderate concentrations of glucose (0.5%) to sporulation medium did not inhibit transcription of two key activators of sporulation, IME1 and IME2, but did increase levels of Sic1p, a cyclin-dependent kinase inhibitor, resulting in a block to meiotic DNA replication. The effects of glucose on Sic1p levels and DNA replication required Grr1p, a component of the SCF(Grr1p) ubiquitin ligase. Sic1p is negatively regulated by Ime2p kinase, and several observations indicate that glucose inhibits meiotic DNA replication through SCF(Grr1p)-mediated destruction of this kinase. First, Ime2p was destabilized in the presence of glucose, and this turnover required Grr1p, a second component of SCF(Grr1p), Cdc53p, and an SCF(Grr1p)-associated E2 enzyme, Cdc34p. Second, Ime2p-ubiquitin conjugates were detected under conditions of rapid Ime2p turnover, and conjugation of Ime2p to ubiquitin required GRR1. Third, a mutant form of Ime2p (Ime2(DeltaPEST)), in which a putative Grr1p-interacting sequence was deleted, was more stable than wild-type Ime2p. Finally, expression of the IME2(DeltaPEST) allele bypassed the block to meiotic DNA replication caused by 0.5% glucose. In addition, Grr1p is required for later events in sporulation independently of its role in Ime2p turnover.

Cell size and Cln-Cdc28 complexes mediate entry into meiosis by modulating cell growth.

In the yeast Saccharomyces cerevisiae, mitotic cell cycle progression depends upon the G(1)-phase cyclin-dependent kinase Cln-Cdc28 and cell growth to a minimum cell size. In contrast, Cln-Cdc28 inhibits entry into meiosis, and a cell growth requirement for sporulation has not been established. Here, we report that entry into meiosis also depends upon cell growth. Moreover, sporulation and cell growth rates were proportional to cell size; large cells grew rapidly and sporulated sooner while smaller cells grew slowly and sporulated later. In addition, Cln2 protein levels were higher in smaller cells suggesting that Cln-Cdc28 activity represses meiosis in smaller cells by preventing cell growth. In support of this hypothesis, loss of Clns, or the presence of a cdc28 mutation increased cell growth specifically in smaller cells and accelerated meiosis in these cells. Finally, overexpression of CLNs repressed meiosis in smaller cells, but not in large cells. Taken together, these results demonstrate that Cln-Cdc28 represses entry into meiosis in part by inhibiting cell growth.

Ime2p and Cdc28p: co-pilots driving meiotic development.

Meiosis can be considered an elaboration of the cell division cycle in the sense that meiosis combines cell-cycle processes with programs specific to meiosis. Each phase of the cell division cycle is driven forward by cell-cycle kinases (Cdk) and coordinated with other phases of the cycle through checkpoint functions. Meiotic differentiation is also controlled by these two types of regulation; however, recent study in the budding yeast S. cerevisiae indicates that progression of meiosis is also controlled by a master regulator specific to meiosis, namely the Ime2p kinase. Below, I describe the overlapping roles of Ime2p and Cdk during meiosis in yeast and speculate on how these two kinases cooperate to drive the progression of meiosis.

Site-specific genomic (SSG) and random domain-localized (RDL) mutagenesis in yeast.

BACKGROUND: A valuable weapon in the arsenal available to yeast geneticists is the ability to introduce specific mutations into yeast genome. In particular, methods have been developed to introduce deletions into the yeast genome using PCR fragments. These methods are highly efficient because they do not require cloning in plasmids. RESULTS: We have modified the existing method for introducing deletions in the yeast (S. cerevisiae) genome using PCR fragments in order to target point mutations to this genome. We describe two PCR-based methods for directing point mutations into the yeast genome such that the final product contains no other disruptions. In the first method, site-specific genomic (SSG) mutagenesis, a specific point mutation is targeted into the genome. In the second method, random domain-localized (RDL) mutagenesis, a mutation is introduced at random within a specific domain of a gene. Both methods require two sequential transformations, the first transformation integrates the URA3 marker into the targeted locus, and the second transformation replaces URA3 with a PCR fragment containing one or a few mutations. This PCR fragment is synthesized using a primer containing a mutation (SSG mutagenesis) or is synthesized by error-prone PCR (RDL mutagenesis). In SSG mutagenesis, mutations that are proximal to the URA3 site are incorporated at higher frequencies than distal mutations, however mutations can be introduced efficiently at distances of at least 500 bp from the URA3 insertion. In RDL mutagenesis, to ensure that incorporation of mutations occurs at approximately equal frequencies throughout the targeted region, this region is deleted at the same time URA3 is integrated. CONCLUSION: SSG and RDL mutagenesis allow point mutations to be easily and efficiently incorporated into the yeast genome without disrupting the native locus.

Signal pathway integration in the switch from the mitotic cell cycle to meiosis in yeast.

Diploid yeast, like most eukaryotes, can undergo meiotic differentiation to form haploid gametes. Meiotic differentiation and cell growth (proliferation) are mutually exclusive programs, and in yeast the switch between growth and meiosis is controlled by nutritional signals. The signaling pathways that mediate nutritional controls on meiotic initiation fall into three broad classes: those that respond to nutrient starvation, those that respond to non-fermentable carbon sources, and those that respond to glucose. At the onset of meiosis, nutritional signaling pathways converge on transcriptional regulation of two genes: IME1, which encodes a transcription factor; and IME2, which encodes a protein kinase. Transcription of IME1 and IME2 trigger initiation of meiosis, and the expression of these two genes is linked with one other, with expression of later meiotic genes and with early meiotic events such as DNA replication. In addition, the signaling pathways that control IME1 and IME2 expression are themselves integrated through a variety of mechanisms. Thus the signal network that controls the switch from growth to meiotic differentiation provides a signaling code that translates different combinations of extracellular signals into appropriate cellular responses.