Probiotic prophylaxis in predicted severe acute pancreatitis: a randomised, double-blind, placebo-controlled trial. Commentary

Rise and fall

https://backend.orbit.dtu.dk/ws/files/189263285/1_s2.0_S1096717619301533_main.pdf

The emergence of adaptive laboratory evolution as an efficient tool for biological discovery and industrial biotechnology.

Multiplexed CRISPR technologies, in which numerous gRNAs or Cas enzymes are expressed at once, have facilitated powerful biological engineering applications, vastly enhancing the scope and efficiencies of genetic editing and transcriptional regulation. In this review, we discuss multiplexed CRISPR technologies and describe methods for the assembly, expression and processing of synthetic guide RNA arrays in vivo. Applications that benefit from multiplexed CRISPR technologies, including cellular recorders, genetic circuits, biosensors, combinatorial genetic perturbations, large-scale genome engineering and the rewiring of metabolic pathways, are highlighted. We also offer a glimpse of emerging challenges and emphasize experimental considerations for future studies.

/pdf/multiplexed-crispr-technologies-for-gene-editing-and-3cvs0mh9ts.pdf

Multiplexed CRISPR technologies for gene editing and transcriptional regulation.

Salmonella Typhimurium establishes systemic infection by replicating in host macrophages Here we show that macrophages infected with S Typhimurium exhibit upregulated glycolysis and decreased serine synthesis, leading to accumulation of glycolytic intermediates The effects on serine synthesis are mediated by bacterial protein SopE2, a type III secretion system (T3SS) effector encoded in pathogenicity island SPI-1 The changes in host metabolism promote intracellular replication of S Typhimurium via two mechanisms: decreased glucose levels lead to upregulated bacterial uptake of 2- and 3-phosphoglycerate and phosphoenolpyruvate (carbon sources), while increased pyruvate and lactate levels induce upregulation of another pathogenicity island, SPI-2, known to encode virulence factors Pharmacological or genetic inhibition of host glycolysis, activation of host serine synthesis, or deletion of either the bacterial transport or signal sensor systems for those host glycolytic intermediates impairs S Typhimurium replication or virulence Salmonella Typhimurium establishes systemic infection by replicating in host macrophages Here, Jiang et al show that infected macrophages exhibit upregulated glycolysis and decreased serine synthesis, leading to accumulation of glycolytic intermediates that promote intracellular replication and virulence of S Typhimurium

/pdf/salmonella-typhimurium-reprograms-macrophage-metabolism-via-30t49kc7nh.pdf

Salmonella Typhimurium reprograms macrophage metabolism via T3SS effector SopE2 to promote intracellular replication and virulence

Along with functional advances in the use of CRISPR/Cas9 for genome editing, endonuclease-deficient Cas9 (dCas9) has provided a versatile molecular tool for exploring gene functions. In principle, differences in cell phenotypes that result from the RNA-guided modulation of transcription levels by dCas9 are critical for inferring with gene function; however, the effect of intracellular dCas9 expression on bacterial morphology has not been systematically elucidated. Here, we observed unexpected morphological changes in Escherichia coli mediated by dCas9, which were then characterized using RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq). Growth rates were severely decreased, to approximately 50% of those of wild type cells, depending on the expression levels of dCas9. Cell shape was changed to abnormal filamentous morphology, indicating that dCas9 affects bacterial cell division. RNA-Seq revealed that 574 genes were differentially transcribed in the presence of high expressi...

High-Level dCas9 Expression Induces Abnormal Cell Morphology in Escherichia coli

Synthetic biology aims to design and construct bacterial genomes harboring the minimum number of genes required for self-replicable life. However, the genome-reduced bacteria often show impaired growth under laboratory conditions that cannot be understood based on the removed genes. The unexpected phenotypes highlight our limited understanding of bacterial genomes. Here, we deploy adaptive laboratory evolution (ALE) to re-optimize growth performance of a genome-reduced strain. The basis for suboptimal growth is the imbalanced metabolism that is rewired during ALE. The metabolic rewiring is globally orchestrated by mutations in rpoD altering promoter binding of RNA polymerase. Lastly, the evolved strain has no translational buffering capacity, enabling effective translation of abundant mRNAs. Multi-omic analysis of the evolved strain reveals transcriptome- and translatome-wide remodeling that orchestrate metabolism and growth. These results reveal that failure of prediction may not be associated with understanding individual genes, but rather from insufficient understanding of the strain's systems biology.

/pdf/adaptive-laboratory-evolution-of-a-genome-reduced-19uf18j6us.pdf

Adaptive laboratory evolution of a genome-reduced Escherichia coli

Microbial cells are versatile hosts for the production of value-added products due to the well-established background knowledge, various genetic tools, and ease of manipulation. Despite those advantages, efficiency of newly incorporated synthetic pathways in microbial cells is frequently limited by innate metabolism, product toxicity, and growth-mediated genetic instability. To overcome those obstacles, a minimal genome harboring only the essential set of genes was proposed, which is a fascinating concept with potential for use as a platform strain. Here, we review the currently available artificial reduced genomes and discuss the prospects for extending use of the genome-reduced strains as programmable chasses. The genome-reduced strains generally showed comparable growth to and higher productivity than their ancestral strains. In Escherichia coli, about 300 genes are estimated as the minimal number of genes under laboratory conditions. However, recent advances revealed that there are non-essential components in essential genes, suggesting that the design principle of minimal genomes should be reconstructed. Current technology is not efficient enough to reduce large amount of interspaced genomic regions or to synthesize the genome. Furthermore, construction of minimal genome frequently has failed due to lack of genomic information. Technological breakthroughs and intense systematic studies on genomes remain tasks.

Minimal genome: Worthwhile or worthless efforts toward being smaller?

Staphylococcus aureus infection is a rising public health care threat. S. aureus is believed to have elaborate regulatory networks that orchestrate its virulence. Despite its importance, the systematic understanding of the transcriptional landscape of S. aureus is limited. Here, we describe the primary transcriptome landscape of an epidemic USA300 isolate of community-acquired methicillin-resistant S. aureus. We experimentally determined 1,861 transcription start sites with their principal promoter elements, including well-conserved -35 and -10 elements and weakly conserved -16 element and 5' untranslated regions containing AG-rich Shine-Dalgarno sequence. In addition, we identified 225 genes whose transcription was initiated from multiple transcription start sites, suggesting potential regulatory functions at transcription level. Along with the transcription unit architecture derived by integrating the primary transcriptome analysis with operon prediction, the measurement of differential gene expression revealed the regulatory framework of the virulence regulator Agr, the SarA-family transcriptional regulators, and β-lactam resistance regulators. Interestingly, we observed a complex interplay between virulence regulation, β-lactam resistance, and metabolism, suggesting a possible tradeoff between pathogenesis and drug resistance in the USA300 strain. Our results provide platform resource for the location of transcription initiation and an in-depth understanding of transcriptional regulation of pathogenesis, virulence, and antibiotic resistance in S. aureus.

/pdf/genome-scale-analysis-of-methicillin-resistant-2ffnxgmof6.pdf

Genome-scale analysis of Methicillin-resistant Staphylococcus aureus USA300 reveals a tradeoff between pathogenesis and drug resistance

Microbial diversity and complexity pose challenges in understanding the voluminous genetic information produced from whole-genome sequences, bioinformatics and high-throughput ‘-omics’ research. These challenges can be overcome by a core blueprint of a genome drawn with a minimal gene set, which is essential for life. Systems biology and large-scale gene inactivation studies have estimated the number of essential genes to be ∼300–500 in many microbial genomes. On the basis of the essential gene set information, minimal-genome strains have been generated using sophisticated genome engineering techniques, such as genome reduction and chemical genome synthesis. Current size-reduced genomes are not perfect minimal genomes, but chemically synthesized genomes have just been constructed. Some minimal genomes provide various desirable functions for bioindustry, such as improved genome stability, increased transformation efficacy and improved production of biomaterials. The minimal genome as a chassis genome for synthetic biology can be used to construct custom-designed genomes for various practical and industrial applications.

Donghui Choe

Papers

High-Level dCas9 Expression Induces Abnormal Cell Morphology in Escherichia coli

Adaptive laboratory evolution of a genome-reduced Escherichia coli

Minimal genome: Worthwhile or worthless efforts toward being smaller?

Genome-scale analysis of Methicillin-resistant Staphylococcus aureus USA300 reveals a tradeoff between pathogenesis and drug resistance

Construction of a minimal genome as a chassis for synthetic biology.