Biogeographical survey of soil microbiomes across sub-Saharan Africa: structure, drivers, and predicted climate-driven changes.

BACKGROUND: Top-soil microbiomes make a vital contribution to the Earth's ecology and harbor an extraordinarily high biodiversity. They are also key players in many ecosystem services, particularly in arid regions of the globe such as the African continent. While several recent studies have documented patterns in global soil microbial ecology, these are largely biased towards widely studied regions and rely on models to interpolate the microbial diversity of other regions where there is low data coverage. This is the case for sub-Saharan Africa, where the number of regional microbial studies is very low in comparison to other continents. RESULTS: The aim of this study was to conduct an extensive biogeographical survey of sub-Saharan Africa's top-soil microbiomes, with a specific focus on investigating the environmental drivers of microbial ecology across the region. In this study, we sampled 810 sample sites across 9 sub-Saharan African countries and used taxonomic barcoding to profile the microbial ecology of these regions. Our results showed that the sub-Saharan nations included in the study harbor qualitatively distinguishable soil microbiomes. In addition, using soil chemistry and climatic data extracted from the same sites, we demonstrated that the top-soil microbiome is shaped by a broad range of environmental factors, most notably pH, precipitation, and temperature. Through the use of structural equation modeling, we also developed a model to predict how soil microbial biodiversity in sub-Saharan Africa might be affected by future climate change scenarios. This model predicted that the soil microbial biodiversity of countries such as Kenya will be negatively affected by increased temperatures and decreased precipitation, while the fungal biodiversity of Benin will benefit from the increase in annual precipitation. CONCLUSION: This study represents the most extensive biogeographical survey of sub-Saharan top-soil microbiomes to date. Importantly, this study has allowed us to identify countries in sub-Saharan Africa that might be particularly vulnerable to losses in soil microbial ecology and productivity due to climate change. Considering the reliance of many economies in the region on rain-fed agriculture, this study provides crucial information to support conservation efforts in the countries that will be most heavily impacted by climate change. Video Abstract.

Kinetics of mutation induction by ultraviolet light in excision-deficient yeast.

We have measured the frequency of UV-induced reversions (locus plus suppressor) for the ochre alleles ade2-1 and lys2-1 and forward mutations (ade2 adex double auxotrophs) in an excision-deficient strain of Saccharomyces cerevisiae (rad2-20). For very low UV doses, both mutational systems exhibit linear induction kinetics. However, as the dose increases, a strikingly different response is observed: in the selective reversion system a transition to higher order induction kinetics occurs near 9 ergs/mm2 (25% survival), whereas in the nonselective forward system the mutation frequency passes through a maximum near 14 ergs/mm2 (4.4% survival) and then declines. This contrast in kinetics cannot be explained in any straightforward way by current models of induced mutagenesis, which have been developed primarily on the basis of bacterial data. The bacterial models are designed to accommodate the quadratic induction kinetics that are frequently observed in these systems. We have derived a mathematical expression for mutation frequency that enables us to fit both the forward and reversion data on the assumptions that mutagenesis is basically a "single event" Poisson process, and that mutation and killing are not necessarily independent of one another. In particular, the dose-response relations are consistent with the idea that the sensitivity of the revertants is about 25% less than that of the original cell population, whereas the sensitivity of the forward mutants is about 29% greater than the population average. We argue that this relatively small differential sensitivity of mutant and nonmutant cells is associated with events that take place during mutation expression and clonal growth.

Heteroduplex repair as an intermediate step of UV mutagenesis in yeast.

We have measured UV-induced mutation frequencies in yeast in a forward, nonselective assay system by scoring white adex ade2 double auxotrophs among parental red-pigmented ade2 clones. The frequencies of sectored and pure mutant clones were determined separately. In excision-defective strains carrying the genes rad1-1, rad3-2 and rad4-4, as well as in the double mutants, rad 1-1 rad 3-2 and rad 1-1 rad 4-4, considerably more sectored than pure clones are induced in the low-dose range; in repair-competent strains, pure mutant clones substantially outnumber the sectored clones. These results can be explained on the basis of known differences in the timing of error-prone repair during the cell division cycle; that is, we assume that error-prone repair occurs primarily before replication in RAD wild-type strains but after replication in excision-deficient mutants. It has been suggested that excision deficiency has a pleiotropic effect on heteroduplex repair and nucleotide excision repair; however, the high percentage (36.6%) of half-sectored clones found in the rad1-1 strain is hard to reconcile with this hypothesis. We propose that heteroduplex repair occurs subsequent to error-prone repair in both excision-proficient and excision-deficient strains.

Control of endoreduplication domains in the Drosophila gut by the knirps and knirps-related genes.

Endoreduplication cycles that lead to an increase of DNA ploidy and cell size occur in distinct spatial and temporal patterns during Drosophila development. Only little is known about the regulation of these modified cell cycles. We have investigated fore- and hindgut development and we present evidence that the Drosophila knirps and knirps-related genes are key components to spatially restrict endoreduplication domains. Our lack and gain-of-function experiments show that knirps and knirps-related which encode nuclear orphan receptors transcriptionally repress S-phase genes of the cell cycle required for DNA replication and that this down-regulation is crucial for gut morphogenesis. Furthermore, we demonstrate that both genes are activated in overlapping expression domains in the fore- and hindgut in response to Wingless and Hedgehog activities emanating from epithelial signaling centers that control the regionalization of the gut tube. Our results provide a novel link between morphogen-dependent positional information and the spatio-temporal regulation of cell cycle activity in the gut.

The influence of surface soil physicochemistry on the edaphic bacterial communities in contrasting terrain types of the Central Namib Desert.

Notwithstanding, the severe environmental conditions, deserts harbour a high diversity of adapted micro-organisms. In such oligotrophic environments, soil physicochemical characteristics play an important role in shaping indigenous microbial communities. This study investigates the edaphic bacterial communities of three contrasting desert terrain types (gravel plains, sand dunes and ephemeral rivers) with different surface geologies in the Central Namib Desert. For each site, we evaluated surface soil physicochemistries and used explorative T-RFLP methodology to get an indication of bacterial community diversities. While grain size was an important parameter in separating the three terrain types physicochemically and specific surface soil types could be distinguished, the desert edaphic bacterial communities displayed a high level of local spatial heterogeneity. Ten variables contributed significantly (P < 0.05) to the variance in the T-RFLP data sets: fine silt, medium and fine sand content, pH, S, Na, Zn, Al, V and Fe concentrations, and 40% of the total variance could be explained by these constraining variables. The results suggest that local physicochemical conditions play a significant role in shaping the bacterial structures in the Central Namib Desert and stress the importance of recording a wide variety of environmental descriptors to comprehensively assess the role of edaphic parameters in shaping microbial communities.

Inducibility of error-prone DNA repair in yeast?

Whereas some experimental evidence suggests that mutagenesis in yeast after treatment with DNA-damaging agents involves inducible functions, a general-acting error-prone repair activity analogous to the SOS system of Escherichia coli has not yet been demonstrated. The current literature on the problem of inducibility of mutagenic repair in yeast is reviewed with emphasis on the differences in the experimental procedures applied.

Quantitative measures of mutagenicity and mutability based on mutant yield data.

We described how mutant yield data (mutants per cell treated) can be used both to compare the mutagenicity of different mutagens, and to characterize the mutability of different cell types. Yield curves reveal the net effect of the lethal and genetic actions of mutagens on cells. Normally, yields are the quantities measured in assays for mutagenesis, and rectilinear plots of such data baldly reveal the amount of experimental error and the extent of actual mutant induction above the background level. Plots of yield versus lethal hits can be used to quantify the relative mutagenic efficiency (RME) of agents whose physical exposure doses otherwise would be incommensurable, as well as the relative mutability (Rmt) of different strains to the same mutagen. Plots of yield versus log dose provide an unambiguous way of assessing the relative mutational sensitivities (Rms) and mutational resolutions (Rmr) of different strains against a given mutagen. Such analysis is important for evaluation of the relative merits of excision-proficient and excision-deficient strains of the same organism as mutagen-testing systems. The mathematical approach outlined here is applied, by way of example, to measurements of UV and 4-NQO induced mutagenesis in both repair-deficient and repair-proficient haploid strains of the yeast Saccharomyces cerevisiae.

Induction of pure and sectored mutant clones in excision-proficient and deficient strains of yeast.

We have found that UV-induced mutation frequency in a forward non-selective assay system (scoring white adex ade2 double auxotroph mutants among the red pigmented ade2 clones) increases linearly with dose up to a maximum frequency of about 3 X 10(-3) mutants per survivor and then declines in both RAD wild-type and rad2 excision deficient strains of Saccharomyces cerevisiae. Mutation frequencies of the RAD and the rad2 strains plotted against survival are nearly identical over the entire survival range. On this basis we conclude that unexcised pyrimidine dimers are the predominant type of pre-mutational lesions in both strains. In the RAD wild-type strain pure mutant clones outnumber sectors in a 10:1 ratio at all doses used; in rad2 this ratio varies from 1:1 at low doses up to 10:1 at high doses. As others have concluded for wild-type strains we find also in the rad2 strain that pure clone formation cannot be accounted for quantitatively by lethal sectoring events alone. We conclude that heteroduplex repair is a crucial step in pure mutant clone formation and we examine the plausibility of certain macromolecular mechanisms according to which heteroduplex repair may be coupled with replication, repair and sister strand exchange in yeast mutagenesis.

Isolation of glutathione-deficient mutants of the yeast Saccharomyces cerevisiae.

Glutathione-deficient (gsh-) mutants of the yeast Saccharomyces cerevisiae were isolated after UV treatment using MNNG as selective agent. For genetic and biochemical characterization 5 mutant strains were chosen which exhibited considerably decreased residual GSH contents varying from 2 to 6% of the wild-type levels. All 5 isolates showed a 2:2 segregation of the gsh-:GSH+ phenotypes alluding to a monogenic recessive mutation. Complementation analysis indicates that all gsh- mutants belong to one complementation group.

Analysis of non-linearities in frequency curves for UV-induced mitotic recombination in wild-type and excision-repair-deficient strains of yeast.

Frequency curves for UV-induced mitotic recombination often are linear at low doses. As dose increases, these curves either increase at higher powers of dose and/or reach a maximum induced frequency and then decline. Similar dose-response patterns have been observed previously for mutation. The non-linearities can arise from higher order effects inherent in the molecular mechanisms of mutagenesis and/or from 'delta-effects' (Eckardt and Haynes, 1977a), i.e., differential probabilities of clone formation for mutant and non-mutant cells. Previously, we have shown that one can distinguish between these two possibilities by plotting the ratio of the induced mutant yield to the linear component of frequency as a function of dose (Haynes et al., 1985). In this study, we have used this ratio, a quantity we call 'apparent survival', to analyse the non-linear regions of the dose-response curves for UV-induced mitotic crossing-over and gene conversion in wild-type (RAD) and excision-repair-deficient (rad3) strains of yeast. Plots of apparent survival versus dose reveal the existence of a positive, non-linear component associated with UV-induced gene conversion in RAD, but not rad3, cells. A high dose decline in frequency, which is observed for UV-induced recombination in both strains, can be attributed to delta-effects.

Analysis of non-linearities in mutation frequency curves.

Mutation frequency curves for ultraviolet light and other mutagens often exhibit non-linear, as well as linear components. A common pattern observed for UV-induced reversion of auxotrophy in yeast is a biphasic, linear-quadratic (or higher order) response. The non-linear component in such a biphasic frequency curve can arise in two distinct, but non-mutually exclusive, ways: (i) as a result of the existence of two-hit processes in the molecular mechanism(s) of mutagenesis; and (ii) as a result of the possible stochastic dependence of mutation and killing, such that the probability of clone formation by the mutant cells differs from that of the non-mutant cells in the population. We describe here a simple mathematical method for distinguishing between these two sources of non-linearity. It is based on the calculation of a quantity that we call 'apparent survival.' This is given, for any mutagen dose chi, by the ratio of the mutant yield to the corresponding linear component of mutation frequency. If the apparent survival rises to values greater than unity before declining at high doses, then there must exist positive two-hit (or higher order) components in the mutational mechanism. If the final slope of the apparent survival curve differs from that of the measured survival curve, then there also exists some degree of stochastic dependence between mutation and killing.

Rat hepatic vinyl chloride metabolites induce gene conversion in the yeast strain D7RAD in vitro and in vivo.

We have tested the genetic activity of gaseous vinyl chloride in vitro and in vivo using the gene-conversion system (trp5-12/trp5-27 leads to TRP+) in the yeast strain D7RAD. To induce, in vitro, TRP+ convertants with 2.5% gaseous vinyl chloride, a rat-liver microsomal system for metabolic activation of the vinyl chloride and dividing yeast cells are required. Neither a deficiency in excision repair (rad3) nor in the error-prone repair pathway (rad6) increased the vinyl-chloride-induced conversion frequencies compared with the repair-competent D7RAD strain. When logarithmically growing cells of the D7RAD strain were injected intravenously into male Wistar rats which inhaled 1% vinyl chloride in air for 24 h, a significant enhancement of the TRP+ conversion frequencies was found compared with that in cells re-isolated from untreated rats. These results indicate that vinyl chloride metabolites from the metabolizing hepatocytes diffuse into yeast cells, which accumulate in the liver capillaries. This supports the hypothesis that the endothelial cells of the liver sinuses, which have hardly any metabolic activity, but give rise to vinyl-chloride-induced hemangiotheliomas (rare type of liver tumor), are transformed by diffusible metabolites of the procarcinogen vinyl chloride.

Analysis of mutagenic DNA repair in a thermoconditional mutant of Saccharomyces cerevisiae. III. Dose-response pattern of mutation induction in UV-irradiated rev2ts cells.

Recent studies regarding the influence of cycloheximide on the temperature-dependent increase in survival and mutation frequencies of a thermoconditional rev2 mutant lead to the suggestion that the REV2-coded mutagenic repair function is UV-inducible. In the present study we show that stationary-phase rev2ts cells are characterized by a biphasic linear-quadratic dose-dependence of mutation induction ("mutation kinetics") of ochre alleles at 23 degrees C (permissive temperature) but linear kinetics at the restrictive temperature of 36 degrees C. Mathematical analysis using a model based on Poisson statistics and a further mathematical procedure, the calculation of "apparent survival", support the assumption that the quadratic component of the reverse mutation kinetics investigated can be attributed to a UV-inducible component of mutagenic DNA repair controlled by the REV2 gene.

Indications for an inducible component of error-prone DNA repair in yeast.

In a thermoconditional mutant of mutagenic DNA repair (rev 2ts = rad 5-8) of Saccharomyces cerevisiae recovery of survival and mutation frequencies can be monitored by incubating UV-irradiated cells in growth medium at a permissive temperature (23 degrees C) before plating and a shift to restrictive temperature (36 degrees C). Inhibition of protein synthesis with cycloheximide during incubation at permissive conditions blocks this REV 2 dependent recovery process in stationary phase rev 2ts cells, whereas it can be reduced but not totally abolished in exponentially growing cells. These results indicate a strict dependence on post-irradiation protein synthesis in stationary phase cells and argue for a considerable constitutive level and only limited inducibility in logarithmic phase cells. The UV inducibility of the REV 2 coded function in stationary phase cells could be confirmed by analysis of the dose-response pattern of the his 5-2 reversion: in stationary phase rev 2ts cells, the quadratic component of the biphasic linear-quadratic induction kinetics found at 23 degrees C, which is interpreted as the consequence of induction of mutagenic repair, is eliminated at 36 degrees C.

Mutagen testing with yeast.

This article deals primarily with the practical aspects of mutagen testing with yeast. Equipment necessary for a laboratory where mutagen testing with yeast is performed, and the most commonly used media, are listed. Some general procedures are described and, finally, for those who have little experience with work of this kind, a precise protocol is given for an experiment with stationary phase cells of the strain D7 of Saccharomyces cerevisiae using the heteroallelic ade2 system as the genetic endpoint. Some experimental data were obtained by students following this protocol using the direct-acting mutagen ethyl methanesulfonate (EMS); these data are discussed and analyzed. More details on the various genetic endpoints available in numerous yeast strains and on the interpretation of dose-dependence data, as well as an extended list of yeast literature, can be found in an article by Eckardt and von Borstel in this volume. Further technical advice is provided in our references to Zimmermann (1975), von Borstel (1981), and Zimmermann et al. (1984).

Analysis of mutagenic DNA repair in a thermoconditional mutant of Saccharomyces cerevisiae. IV. Influence of DNA replication and excision repair on REV2 dependent UV-mutagenesis and repair.

A double mutant being thermoconditionally defective in mutation induction as well as in repair of pre-lethal UV-induced DNA damage (rev2ts) and deficient in excision repair (rad3-2) was studied in temperature-shift experiments. The influence of inhibitors of DNA replication (hydroxyurea, aphidicolin) was determined. Additionally, an analysis of the dose-response pattern of mutation induction ("mutation kinetics") at several ochre alleles was carried out. It was concluded that the UV-inducible REV2 dependent mutagenic repair process is not induced in excision-deficient cells. In excision-deficient cells, REV2 dependent mutation fixation is slow and mostly post-replicative though not dependent on DNA replication. The REV2 mediated mutagenic process could be separated from the repair function.

Analysis of mutagenic DNA repair in a thermoconditional repair mutant of Saccharomyces cerevisiae. I. Influence of cycloheximide on UV-irradiated stationary phase rev2ts cells.

Using the thermoconditional yeast mutant rev2ts that controls an apparently site-specific step of mutagenic DNA repair it was possible to measure the time course of REV2 dependent UV-induced reversion of the ochre allele his5-2 and recovery of survival for UV-treated stationary phase cells: due to the rev2ts coded protein being active at 23 degrees C, survival and mutation frequencies increased with duration of incubation under permissive conditions in growth medium before the temperature was shifted to 36 degrees C (restrictive temperature). This increase was abolished in the presence of the protein synthesis inhibitor, cycloheximide. Furthermore, the REV2 dependent recovery of survival could be blocked or nearly blocked by cycloheximide added at any time during repair. Therefore, REV2 dependent repair can be characterized as a process requiring concomitant protein synthesis. These findings give further support to the concept that in yeast, mutagenesis involves UV inducible components of DNA repair.

Analysis of mutagenic DNA repair in a thermoconditional repair mutant of Saccharomyces cerevisiae. II. Influence of cycloheximide on UV-irradiated exponentially growing rev2ts cells.

The time course of REV2 dependent recovery from prelethal UV damage and UV-induced locus-specific reversion of the his5-2 allele was determined in temperature-shift experiments by use of a thermoconditional allele of the rev2 gene (rad5-8, rev2ts). In UV-irradiated, exponentially growing rev2ts cells the REV2 dependent repair activity persists for up to 8 h at permissive temperature (23 degrees C), while the REV2 dependent mutagenic process is mostly completed within 2 h. The REV2 dependent process in exponentially growing cells is highly impaired by inhibition of protein synthesis. However, a REV2 dependent repair activity independent of de novo synthesis is detectable, even in the presence of up to 200 micrograms/ml cycloheximide, a response not found in stationary phase cells. Thus, the REV2 dependent process seems to be partially constitutive in exponentially growing cells. Additionally, exponentially growing rev2ts cells were considerably more UV-sensitive at restrictive temperature (36 degrees C) than were stationary phase cells.