Mutational hotspots lead to robust but suboptimal adaptive outcomes in certain environments.

The observed mutational spectrum of adaptive outcomes can be constrained by many factors. For example, mutational biases can narrow the observed spectrum by increasing the rate of mutation at isolated sites in the genome. In contrast, complex environments can shift the observed spectrum by defining fitness consequences of mutational routes. We investigate the impact of different nutrient environments on the evolution of motility in Pseudomonas fluorescens Pf0-2x (an engineered non-motile derivative of Pf0-1) in the presence and absence of a strong mutational hotspot. Previous work has shown that this mutational hotspot can be built and broken via six silent mutations, which provide rapid access to a mutation that rescues swimming motility and confers the strongest swimming phenotype in specific environments. Here, we evolved a hotspot and non-hotspot variant strain of Pf0-2x for motility under nutrient-rich (LB) and nutrient-limiting (M9) environmental conditions. We observed the hotspot strain consistently evolved faster across all environmental conditions and its mutational spectrum was robust to environmental differences. However, the non-hotspot strain had a distinct mutational spectrum that changed depending on the nutrient environment. Interestingly, while alternative adaptive mutations in nutrient-rich environments were equal to, or less effective than, the hotspot mutation, the majority of these mutations in nutrient-limited conditions produced superior swimmers. Our competition experiments mirrored these findings, underscoring the role of environment in defining both the mutational spectrum and the associated phenotype strength. This indicates that while mutational hotspots working in concert with natural selection can speed up access to robust adaptive mutations (which can provide a competitive advantage in evolving populations), they can limit exploration of the mutational landscape, restricting access to potentially stronger phenotypes in specific environments.

Different adaptive strategies in E. coli populations evolving under macronutrient limitation and metal ion limitation.

BACKGROUND: Adaptive responses to nutrient limitation involve mutations that increase the efficiency of usage or uptake of the limiting nutrient. However, starvation of different nutrients has contrasting effects on physiology, resulting in different evolutionary responses. Most studies performed to understand these evolutionary responses have focused only on macronutrient limitation. Hence our understanding of adaptation under limitation of other forms of nutrients is limited. In this study, we compared the evolutionary response in populations evolving under growth-limiting conditions for a macronutrient and a major cation. RESULTS: We evolved eight populations of E. coli in nutrient-limited chemostats for 400 generations to identify the genetic basis of the mechanisms involved in efficient usage of two nutrients: nitrogen and magnesium. Our population genomic sequencing work, based on this study and previous work, allowed us to identify targets of selection under these nutrient limiting conditions. Global transcriptional regulators glnGL were targets of selection under nitrogen starvation, while proteins involved in outer-membrane biogenesis (genes from the lpt operon) were targets of selection under magnesium starvation. The protein involved in cell-cycle arrest (yhaV) was a target of selection in both environments. We re-constructed specific mutants to analyze the effect of individual mutations on fitness in nutrient limiting conditions in chemostats and in batch cultures. We further demonstrated that adaptation to nitrogen starvation proceeds via a nutrient specific mechanism, while that to magnesium starvation involves a more general mechanism. CONCLUSIONS: Our results show two different forms of adaptive strategies under limitation of nutrients that effect cellular physiology in different ways. Adaptation to nitrogen starvation proceeds by upregulation of transcriptional regulator glnG and subsequently of transporter protein amtB, both of which results in increased nitrogen scavenging ability of the cell. On the other hand, adaptation to magnesium starvation proceeds via the restructuring of the cell outer-membrane, allowing magnesium to be redistributed to other biological processes. Also, adaptation to the chemostat environment involves selection for loss of function mutations in genes that under nutrient-limiting conditions interfere with continuous growth.

Transcriptomic and Phenotypical Analysis of the Physiological Plasticity of Chamaecyparis hodginsii Roots under Different Nutrient Environments and Adjacent Plant Competition.

Chamaecyparis hodginsii seedlings undergo significant changes during growth due to different nutrient environments and adjacent plant competition, which is evident in the physiological plasticity changes in their roots. Therefore, in this experiment, 20 one-year-old elite C. hodginsii family seedlings were selected as the test objects, and the different nutrient environments and adjacent plant competition environments in nature were artificially simulated. Four nutrient environments (N heterogeneous nutrient environment, P heterogeneous nutrient environment, K heterogeneous nutrient environment, and homogeneous environment) and three planting patterns (single plant, conspecific neighbor, and heterospecific neighbor) were set up to determine the differences in root physiological indexes and plasticity of different family seedlings, and the families and treatment combinations with higher comprehensive evaluation were selected. The transcriptome sequencing of fine roots of C. hodginsii under different treatments was performed to analyze the differentially expressed genes. The results showed that the root activity, antioxidant enzyme activity, and nutrient element content of C. hodginsii seedlings in the N and P heterogeneous environments were higher than those in the homogeneous nutrient environment, while there was no significant difference between the K heterogeneous nutrient environment and the homogeneous environment, but MDA content was higher than that in other nutrient environments. The root activity and antioxidant enzyme activity in the competitive patterns were generally higher than those in the single plant and reached the peak in the heterospecific neighbor. The root physiological plasticity index of line 490 was the highest, but the comprehensive evaluation of root physiological indexes of lines 539 and 535 was better. The pattern with the highest comprehensive evaluation score was P heterogeneous nutrient environment × heterospecific neighbor. The effects of the N and P heterogeneous nutrient environments on root transcriptome genes were similar, which significantly increased DNA transcription and regulatory factor activity, while K heterogeneous nutrient environment focused on the regulation of root enzyme activity. The heterogeneous nutrient environment induces the conduction of hormone signals in the roots of C. hodginsii and induces the synthesis of phenylpropanone. The biosynthesis of phenylpropanone in the roots of C. hodginsii will increase significantly under competitive patterns. In summary, the N and P heterogeneous nutrient environments and the heterospecific neighbor can improve the root physiological indexes of C. hodginsii families, and the root physiological indexes of lines 539 and 535 are the best. The nutrient environment and competition pattern mainly affect the root system to transmit hormone signals to regulate enzyme activity.

Response of the root morphological structure of Fokienia hodginsii seedlings to competition from neighboring plants in a heterogeneous nutrient environment.

Introduction: Critical changes often occur in Fokienia hodginsii seedlings during the process of growth owing to differences in the surrounding environment. The most common differences are heterogeneous nutrient environments and competition from neighboring plants. Methods: In this study, we selected one-year-old, high-quality Fokienia hodginsii seedlings as experimental materials. Three planting patterns were established to simulate different competitive treatments, and seedlings were also exposed to three heterogeneous nutrient environments and a homogeneous nutrient environment (control) to determine their effect on the root morphology and structure of F. hodginsii seedlings. Results: Heterogeneous nutrient environments, compared with a homogeneous environment, significantly increased the dry matter accumulation and root morphology indexes of the root system of F. hodginsii, which proliferated in nutrient-rich patches, and the P heterogeneous environment had the most significant enhancement effect, with dry matter accumulation 70.2%, 7.0%, and 27.0% higher than that in homogeneous and N and K heterogeneous environments, respectively. Homogeneous environments significantly increased the specific root length and root area of the root system; the dry matter mass and morphological structure of the root system of F. hodginsii with a heterospecific neighbor were higher than those under conspecific neighbor and single-plant treatments, and the root area of the root system under the conspecific neighbor treatment was higher than that under the heterospecific neighbor treatment, by 20% and 23%, respectively. Moreover, the root system under heterospecific neighbor treatment had high sensitivity; the heterogeneous nutrient environment increased the mean diameter of the fine roots of the seedlings of F. hodginsii and the diameter of the vascular bundle, and the effect was most significant in the P heterogeneous environment, exceeding that in the N and K heterogeneous environments. The effect was most significant in the P heterogeneous environment, which increased fine root diameter by 20.5% and 10.3%, respectively, compared with the homogeneous environment; in contrast, the fine root vascular ratio was highest in the homogeneous environment, and most of the indicators of the fine root anatomical structure in the nutrient-rich patches were of greater values than those in the nutrient-poor patches in the different heterogeneous environments; competition promoted most of the indicators of the fine root anatomical structure of F. hodginsii seedlings. According a principal component analysis (PCA), the N, Pm and K heterogeneous environments with heterospecific neighbors and the P heterogeneous environment with a conspecific neighbor had higher evaluation in the calculation of eigenvalues of the PCA. Discussion: The root dry matter accumulation, root morphology, and anatomical structure of F. hodginsii seedlings in the heterogeneous nutrient environment were more developed than those in the homogeneous nutrient environment. The effect of the P heterogeneous environment was the most significant. The heterospecific neighbor treatment was more conducive to the expansion and development of root morphology of F. hodginsii seedlings than were the conspecific neighbor and single-plant treatments.

Screening in serum-derived medium reveals differential response to compounds targeting metabolism.

A challenge for screening new anticancer drugs is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels, which can influence cell metabolism and drug sensitivity. A general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To address this, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We screened several small molecule libraries and found that compounds targeting metabolic enzymes were differentially effective in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

Variations of rhizosphere and bulk soil microbial community in successive planting of Chinese fir (Cunninghamia lanceolata).

Successive planting and monoculture, as common forest management methods, are widely used globally, especially in Chinese fir plantations in the subtropical areas of southern China. Although soil fertility depletion and productivity decline caused by successive planting have been widely reported, the underlying mechanism is still ambiguous. In this study, the composition and diversity of soil microorganisms (rhizosphere and bulk soils) in Chinese fir seedlings exposed to successive planting soils (first-generation Chinese fir seedings, FCP. second-generation Chinese fir seedings, SCP. third-generation Chinese fir seedings, TCP) and broadleaf tree species soil (Phoebe zhennan S. Lee et F. N. Wei, CK) were examined with high-throughput sequencing technology. Our findings revealed that the diversity and richness of bacterial and fungal communities were remarkably reduced in TCP than FCP and SCP, and were remarkably different between FCP and SCP. At the phylum level, the fungi with greatest relative abundance were Basidiomycota (5.74-32.88%) and Ascomycota (57.63-87.38%), while the bacteria with the greatest relative abundance were Acidobacteria (23.16-31.17%) and Proteobacteria (24.71-29.32%) for all treatments in both soil types. Additionally, the relative abundance of some pathogens (Penicillium and Burkholderia) was significantly higher in TCP than in FCP and SCP, suggesting that the presence of pathogens is an important factor in increasing the incidence of soil-borne sickness. Moreover, changes in fungal and bacterial communities were predominantly driven by soil dissolved organic carbon (DOC), DOC/DON ratio (DOCN), NO3 --N, microbial biomass carbon (MBC), and MBC/MBN ratio (MBCN). Overall, the long-term monoculture of Chinese fir promotes the microecological imbalance of rhizosphere and bulk soil, and remarkably reduced soil microbial community diversity. These results can provide a scientific support for the implementation of future management measures for fir plantations (e.g., fertilization, addition of microbial fungicides, and construction of mixed forests).

Environmental UVR Levels and Skin Pigmentation Gene Variants Associated with Folate and Homocysteine Levels in an Elderly Cohort.

Ultraviolet radiation (UVR) is a ubiquitous exposure which may contribute to decreased folate levels. Skin pigmentation mediates the biological effect of UVR exposure, but its relationship to folate levels is unexamined. Interactions may exist between UVR and pigmentation genes in determining folate status, which may, in turn, impact homocysteine levels, a potential risk factor for multiple chronic diseases. Therefore, independent and interactive influences of environmental UVR and genetic variants related to skin pigmentation (MC1R-rs1805007, IRF4-rs12203592 and HERC2-rs12913832) on folate (red blood cell (RBC) and serum) and homocysteine levels were examined in an elderly Australian cohort (n = 599). Genotypes were assessed by RT/RFLP-PCR, and UVR exposures were assessed as the accumulated erythemal dose rate accumulated over 4 months (4M-EDR). Multivariate analysis found significant negative associations between 4M-EDR and RBC folate (p < 0.001, ß = -0.19), serum folate (p = 0.045, ß = -0.08) and homocysteine levels (p < 0.001, ß = -0.28). Significant associations between MC1R-rs1805007 and serum folate levels (p = 0.020), and IRF4-rs12203592 and homocysteine levels (p = 0.026) occurred but did not remain significant following corrections with confounders. No interactions between 4M-EDR and pigmentation variants in predicting folate/homocysteine levels were found. UVR levels and skin pigmentation-related variants are potential determinants of folate and homocysteine status, although, associations are mixed and complex, with further studies warranted.

The Soil Nutrient Environment Determines the Strategy by Which Bacillus velezensis HN03 Suppresses Fusarium wilt in Banana Plants.

Biological control agents (BCAs) are considered as one of the most important strategies for controlling Fusarium wilt, and bioorganic fertilizer, in particular, has been extensively investigated. However, little is known regarding how a biocontrol microorganism affects the suppression mechanisms when combined with different amendments. In this study, a pot experiment was performed using banana plants to investigate the different mechanisms by which the biocontrol bacterium Bacillus velezensis HN03 (isolated from our laboratory) and amendments suppress Fusarium wilt. The incidence of banana wilt was decreased under HN03 and was reduced further when HN03 was combined with compost, particularly wormcast. In the suppression of Fusarium wilt, HN03 was found to influence the soil environment in various ways. HN03 increased the peroxidase level, which improves plant defense, and was highest when combined with wormcast, being 69 times higher than when combined with cow dung compost. The high accumulation of Mg and P in the "HN03 + wormcast" and Zn and Mn in the "HN03 + cow dung" treatments was negatively correlated with disease incidence. Furthermore, HN03 re-established the microbial community destroyed by the pathogen and further increased the level of suppression in the wormcast. HN03 also enhanced the functional traits of the soil, including defensive mechanism-related traits, and these traits were further enhanced by the combination of HN03 + wormcast.

Interactions in the (Pre)metastatic Niche Support Metastasis Formation.

Metastasis formation is the leading cause of death in cancer patients. Thus, understanding and targeting this process is an unmet need. Crucial steps during the establishment of metastases include the (pre)metastatic niche formation. This process relies on the interaction of the primary tumor with the environment of distant organs (premetastatic niche) and the interaction of cancer cells with their environment when arriving in a distant organ (metastatic niche). Here, we summarize the current knowledge on the interactions in the tumor environment that result in (pre)metastatic niche formation, specifically in the context of tumor secreted factors, extracellular matrix, immune as well as stromal cells, and nutrient availability. We further highlight strategies to disrupt these interactions as therapeutic interventions against metastases.

The role of metabolism in bacterial persistence.

Bacterial persisters are phenotypic variants with extraordinary tolerances toward antibiotics. Persister survival has been attributed to inhibition of essential cell functions during antibiotic stress, followed by reversal of the process and resumption of growth upon removal of the antibiotic. Metabolism plays a critical role in this process, since it participates in the entry, maintenance, and exit from the persister phenotype. Here, we review the experimental evidence that demonstrates the importance of metabolism to persistence, highlight the successes and potential of targeting metabolism in the search for anti-persister therapies, and discuss the current methods and challenges to understand persister physiology.