The Devil Is in the Microbial Genetic Details.

The work of Zeevi et al. (2019) in a recent issue of Nature shows that variations in gene content and organization between different strains of the same microbial species are widespread in the human gut microbiota and could be linked to many measures of health.

Microbial Genetics: Stress Management.

Microbes constitute the very core of our existence. Long believed to be a nuisance and proponents of various disease, latest research point toward their functions in processes that can prove beneficial for human survival and afford long-term protection from disease. The wide range of functions exhibited by a host of microbes implies diversity and heterogeneity at the level of the molecular machinery, thus stressing the need to take a closer look at the molecular underpinnings that dictate distinct outcomes.

Reliable, scalable functional genetics in bloodstream-form Trypanosoma congolense in vitro and in vivo.

Animal African trypanosomiasis (AAT) is a severe, wasting disease of domestic livestock and diverse wildlife species. The disease in cattle kills millions of animals each year and inflicts a major economic cost on agriculture in sub-Saharan Africa. Cattle AAT is caused predominantly by the protozoan parasites Trypanosoma congolense and T. vivax, but laboratory research on the pathogenic stages of these organisms is severely inhibited by difficulties in making even minor genetic modifications. As a result, many of the important basic questions about the biology of these parasites cannot be addressed. Here we demonstrate that an in vitro culture of the T. congolense genomic reference strain can be modified directly in the bloodstream form reliably and at high efficiency. We describe a parental single marker line that expresses T. congolense-optimized T7 RNA polymerase and Tet repressor and show that minichromosome loci can be used as sites for stable, regulatable transgene expression with low background in non-induced cells. Using these tools, we describe organism-specific constructs for inducible RNA-interference (RNAi) and demonstrate knockdown of multiple essential and non-essential genes. We also show that a minichromosomal site can be exploited to create a stable bloodstream-form line that robustly provides >40,000 independent stable clones per transfection-enabling the production of high-complexity libraries of genome-scale. Finally, we show that modified forms of T. congolense are still infectious, create stable high-bioluminescence lines that can be used in models of AAT, and follow the course of infections in mice by in vivo imaging. These experiments establish a base set of tools to change T. congolense from a technically challenging organism to a routine model for functional genetics and allow us to begin to address some of the fundamental questions about the biology of this important parasite.

An invitation to the marriage of metagenomics and metabolomics.

Metagenomics seeks to characterize the composition of microbial communities, their operations, and their dynamically coevolving relationships with the habitats they occupy without having to culture community members. Uniting metagenomics with analyses of the products of microbial community metabolism (metabolomics) will shed light on how microbial communities function in a variety of environments, including the human body.

Reconstruction of Microbial Haplotypes by Integration of Statistical and Physical Linkage in Scaffolding.

DNA sequencing technologies provide unprecedented opportunities to analyze within-host evolution of microorganism populations. Often, within-host populations are analyzed via pooled sequencing of the population, which contains multiple individuals or "haplotypes." However, current next-generation sequencing instruments, in conjunction with single-molecule barcoded linked-reads, cannot distinguish long haplotypes directly. Computational reconstruction of haplotypes from pooled sequencing has been attempted in virology, bacterial genomics, metagenomics, and human genetics, using algorithms based on either cross-host genetic sharing or within-host genomic reads. Here, we describe PoolHapX, a flexible computational approach that integrates information from both genetic sharing and genomic sequencing. We demonstrated that PoolHapX outperforms state-of-the-art tools tailored to specific organismal systems, and is robust to within-host evolution. Importantly, together with barcoded linked-reads, PoolHapX can infer whole-chromosome-scale haplotypes from 50 pools each containing 12 different haplotypes. By analyzing real data, we uncovered dynamic variations in the evolutionary processes of within-patient HIV populations previously unobserved in single position-based analysis.

Evolutionary Trajectories to Antibiotic Resistance.

The ability to predict the evolutionary trajectories of antibiotic resistance would be of great value in tailoring dosing regimens of antibiotics so as to maximize the duration of their usefulness. Useful prediction of resistance evolution requires information about (a) the mutation supply rate, (b) the level of resistance conferred by the resistance mechanism, (c) the fitness of the antibiotic-resistant mutant bacteria as a function of drug concentration, and (d) the strength of selective pressures. In addition, processes including epistatic interactions and compensatory evolution, coselection of drug resistances, and population bottlenecks and clonal interference can strongly influence resistance evolution and thereby complicate attempts at prediction. Currently, the very limited quantitative data on most of these parameters severely limit attempts to accurately predict trajectories of resistance evolution.

Ranking microbial metabolomic and genomic links in the NPLinker framework using complementary scoring functions.

Specialised metabolites from microbial sources are well-known for their wide range of biomedical applications, particularly as antibiotics. When mining paired genomic and metabolomic data sets for novel specialised metabolites, establishing links between Biosynthetic Gene Clusters (BGCs) and metabolites represents a promising way of finding such novel chemistry. However, due to the lack of detailed biosynthetic knowledge for the majority of predicted BGCs, and the large number of possible combinations, this is not a simple task. This problem is becoming ever more pressing with the increased availability of paired omics data sets. Current tools are not effective at identifying valid links automatically, and manual verification is a considerable bottleneck in natural product research. We demonstrate that using multiple link-scoring functions together makes it easier to prioritise true links relative to others. Based on standardising a commonly used score, we introduce a new, more effective score, and introduce a novel score using an Input-Output Kernel Regression approach. Finally, we present NPLinker, a software framework to link genomic and metabolomic data. Results are verified using publicly available data sets that include validated links.

High temperatures enhance the microbial genetic potential to recycle C and N from necromass in high-mountain soils.

Climate change is strongly affecting high-mountain soils and warming in particular is associated with pronounced changes in microbe-mediated C and N cycling, affecting plant-soil interactions and greenhouse gas balances and therefore feedbacks to global warming. We used shotgun metagenomics to assess changes in microbial community structures, as well as changes in microbial C- and N-cycling potential and stress response genes and we linked these data with changes in soil C and N pools and temperature-dependent measurements of bacterial growth rates. We did so by incubating high-elevation soil from the Swiss Alps at 4°C, 15°C, 25°C, or 35°C for 1 month. We found no shift with increasing temperature in the C-substrate-degrader community towards taxa more capable of degrading recalcitrant organic matter. Conversely, at 35°C, we found an increase in genes associated with the degradation and modification of microbial cell walls, together with high bacterial growth rates. Together, these findings suggest that the rapidly growing high-temperature community is fueled by necromass from heat-sensitive taxa. This interpretation was further supported by a shift in the microbial N-cycling potential towards N mineralization and assimilation under higher temperatures, along with reduced potential for conversions among inorganic N forms. Microbial stress-response genes reacted inconsistently to increasing temperature, suggesting that the high-temperature community was not severely stressed by these conditions. Rather, soil microbes were able to acclimate by changing the thermal properties of membranes and cell walls as indicated by an increase in genes involved in membrane and cell wall modifications as well as a shift in the optimum temperature for bacterial growth towards the treatment temperature. Overall, our results suggest that high temperatures, as they may occur with heat waves under global warming, promote a highly active microbial community capable of rapid mineralization of microbial necromass, which may transiently amplify warming effects.

Elucidating the molecular architecture of adaptation via evolve and resequence experiments.

Evolve and resequence (E&R) experiments use experimental evolution to adapt populations to a novel environment, then next-generation sequencing to analyse genetic changes. They enable molecular evolution to be monitored in real time on a genome-wide scale. Here, we review the field of E&R experiments across diverse systems, ranging from simple non-living RNA to bacteria, yeast and the complex multicellular organism Drosophila melanogaster. We explore how different evolutionary outcomes in these systems are largely consistent with common population genetics principles. Differences in outcomes across systems are largely explained by different starting population sizes, levels of pre-existing genetic variation, recombination rates and adaptive landscapes. We highlight emerging themes and inconsistencies that future experiments must address.

"When worlds collide and smuts converge": Tales from the 1st International Ustilago/Smut Convergence.

From the evening of March 12, till dinner on March 13, 2017, the 1st International Ustilago/Smut Convergence took place as a workshop prior to the start of the 29th Fungal Genetics Conference, in Asilomar, California. The overall goals of the meeting were to expand the smut model systems being used and to expand participation by the next generations of scientists with these fungi. These goals were implemented through a combination of emphasis on student and post-doc presentations, mentoring of such individuals, and active recruitment of participation by groups under-represented at such meetings in recent years in the US, especially those from Latin America and other Spanish-speaking countries. Work was presented at the first workshop on U. maydis, Sporosorium reilianum, Microbotryum violaceum, U. esculenta, and Thecaphora thlaspeos. Students and post-doctoral researchers were encouraged to present their "just-in-time," as-yet-unpublished data, in a safe environment, with the understanding of those attending the meeting that this early access was a privilege not to be taken advantage of. The result was lively and constructive discussion, including a variety of presentations by these young scientists on putative and characterized smut effector proteins, clearly at the forefront of such research, even considering the advances presented later that week at the Fungal Genetics Conference. This review also briefly compares the first meeting with the events of the recent 2nd International Ustilago/Smut Convergence (March 11-12, 2019), which ended with a tribute to Prof. Dr. Regine Kahmann, in honor of her career, and especially for her contributions to the field of smut genetics.

Genetics is the logic of life (at least of mine).

I retrace my path from math to medicine to biochemistry to yeast genetics, my focus on infectious diseases of yeast and finally prions. My discovery of yeast prions relied on my particular focus on the logical relations of non-chromosomal genetic elements and the chromosomal genes involved in their propagation and expression. Pursuing an understanding of yeast prions involved structural biology based on genetics, solid-state NMR, population genetics and more genetics.

Strategies for gene disruption and expression in filamentous fungi.

Filamentous fungi can produce many valuable secondary metabolites; among these fungi, endophytic fungi play an ecological role in mutualistic symbiosis with plants, including promoting plant growth, disease resistance, and stress resistance. However, the biosynthesis of most secondary metabolites remains unclear, and knowledge of the interaction mechanisms between endophytes and plants is still limited, especially for some novel fungi, due to the lack of genetic manipulation tools for novel species. Herein, we review the newly discovered strategies of gene disruption, such as the CRISPR-Cas9 system, the site-specific recombination Cre/loxP system, and the I-SceI endonuclease-mediated system in filamentous fungi. Gene expression systems contain using integration of target genes into the genome, host-dependent expression cassette construction depending on the host, a host-independent, universal expression system independent of the host, and reporter-guided gene expression for filamentous fungi. Furthermore, the Newly CRISPRi, CRISPRa, and the selection markers were also discussed for gene disruption and gene expression were also discussed. These studies lay the foundation for the biosynthesis of secondary metabolites in these organisms and aid in understanding the ecological function of filamentous fungi.

Selectable marker recycling in the nonconventional yeast Xanthophyllomyces dendrorhous by transient expression of Cre on a genetically unstable vector.

Selectable marker recycling is a basic technique in bioengineering. However, this technique is usually unavailable in non-model microorganisms. In this study, we proposed a simple and efficient method for selectable marker recycling in the astaxanthin-synthesizing yeast Xanthophyllomyces dendrorhous. This method was based on a Cre-loxP system, in which the transient expression of the Cre recombinase was controlled by a genetically unstable vector independent of episomal plasmids and inducible promoters. The selectable markers in single-gene locus and multigene loci were removed along with the loss of the Cre vector with a ratio of 100% and 29%, respectively. The significance of the method was highlighted by the finding that stable autotrophic mutants were not readily obtained in X. dendrorhous. Comparative studies in X. dendrorhous and the non-homologous end joining dominant yeast Yarrowia lipolytica suggested that the method could be universally used in homologous recombination dominant yeasts.

Stress tolerance phenotype of industrial yeast: industrial cases, cellular changes, and improvement strategies.

Yeast is widely used in the baking, biocontrol, brewing, and bio manufacturing industries. In the baking industry alone, around two million tons of yeast are consumed worldwide every year. While yeast brings delicious and healthy lives to humans, we find that stress resistance of yeast is essential for the development of bioindustry. Whether during baking, biocontrol, brewing, bio manufacturing, or in other industries, yeast faces a variety of environmental stresses that have a great impact on its activity, transformation ability, etc., which make the production process uncertain. Therefore, robust yeast strains that can resist various environmental and endogenous stresses are needed. In recent years, many studies have investigated the stress resistance of laboratory strains and specific methods to improve stress resistance; however, applying these findings to industrial yeast is difficult. In this paper, based on summarizing the work of predecessors, we put forward the main steps to improve the stress resistance of industrial yeast systematically, which may provide a reference for researchers.

CRISPR-Cas9-assisted native end-joining editing offers a simple strategy for efficient genetic engineering in Escherichia coli.

Unlike eukaryotes, prokaryotes are less proficient in homologous recombination (HR) and non-homologous end-joining (NHEJ). All existing genomic editing methods for Escherichia coli (E. coli) rely on exogenous HR or NHEJ systems to repair DNA double-strand breaks (DSBs). Although an E. coli native end-joining (ENEJ) system has been reported, its potential in genetic engineering has not yet been explored. Here, we present a CRISPR-Cas9-assisted native end-joining editing and show that ENEJ-dependent DNA repair can be used to conduct rapid and efficient deletion of chromosome fragments up to 83 kb or gene inactivation. Moreover, the positive rate and editing efficiency are independent of high-efficiency competent cells. The method requires neither exogenous DNA repair systems nor introduced editing template. The Cas9-sgRNA complex is the only foreign element in this method. This study is the first successful engineering effort to utilize ENEJ mechanism in genomic editing and provides an effective strategy for genetic engineering in bacteria that are inefficient in HR and NHEJ.

New Insights of Ustilago maydis as Yeast Model for Genetic and Biotechnological Research: A Review.

The basidiomycete Ustilago maydis is a biotrophic organism responsible for corn smut disease. In recent years, it has become one of the most promising models for biochemical and biotechnological research due to advantages, such as rapid growth, and easy genetic manipulation. In some aspects, this yeast is more similar to complex eukaryotes, such as humans, compared to standard laboratory yeast models. U. maydis can be employed as a tool to explore physiological processes with more versatility than other fungi. Previously, U. maydis was only considered as a phytopathogenic fungus, but different studies have shown its potential as a research model. Therefore, numerous promising studies have focused on deepening our understanding of the natural interactions, enzyme production, and biotechnological capacity. In this review, we explore general characteristics of U. maydis, both as pathogenic and "innocuous" basidiomycete. Additionally, a comparison with other yeast models focusing on genetic, biochemical, and biotechnological research are analyzed, to emphasize the versatility, dynamism, and novelty that U. maydis has as a research model. In this review, we highlight the applications of the yeast form of the fungus; however, since the filamentous form is also of relevance, it is addressed in the present work, as well.

Heterologous gene expression in the human gut bacteria Eubacterium rectale and Roseburia inulinivorans by means of conjugative plasmids.

Commensal butyrate-producing bacteria in the Firmicutes phylum are abundant in the human intestine and are important for maintaining health. However, understanding of the metabolism and host interaction of these bacteria is limited by the lack of genetic modification techniques. Here we establish a protocol enabling the transfer of autonomously-replicating shuttle vectors by conjugative plasmid transfer from an Escherichia coli donor into representatives of an important sub-group of strictly anaerobic human colonic Firmicutes. Five different plasmid shuttle vectors were tested, each carrying a different origin of replication from Gram-positive bacteria. Plasmid pMTL83151 (pCB102 replicon) were successfully transferred into two strains of Eubacterium rectale, while pMTL83151 and pMTL82151 (pBP1 replicon) were transferred into Roseburia inulinivorans A2-194. Plasmids that carried a Streptococcus bovis JB1 glycoside hydrolase family 16 ß-(1,3-1,4)-glucanase gene were constructed and conjugated into Roseburia inulinivorans A2-194 and Eubacterium rectale T1-815, resulting in successful heterologous expression of this introduced enzymatic activity in these two strains of butyrate-producing Firmicutes.