Directed yeast genome evolution by controlled introduction of trans-chromosomic structural variations

Background: Naturally occurring structural variations (SVs) are a considerable source of genomic variation and can reshape chromosomes 3D architecture. The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system has been proved to generate random SVs to impact phenotypes and thus constitutes powerful drivers of directed genome evolution. However, controllable methods to introduce complex SVs and revealing the related molecular mechanism has so far remained challenging. Results: We develop a SV-prone yeast strain by using SCRaMbLE with two synthetic chromosomes, synV and synX. An heterologous biosynthesis pathway allowing a high throughput screen for increased yield of astaxanthin is used as readout and a proof of concept for the application of SV in industry. We report here that complex SVs, including a pericentric inversion and a trans-chromosomes translocation between synV and synX, result in two neochromosomes and a 2.7-fold yield of astaxanthin. We mapped genetic targets contributing to higher astaxanthin yield and demonstrated the SVs can led to large reorganization of the genetic information along the chromosomes. We also used the model learned from the aforementioned random screen and successfully harnessed the precise introduction of trans-chromosomes translocation and pericentric inversions by rational design. Conclusions: Our work provides an effective tool to not only accelerate the directed genome evolution but also reveal mechanistic insight of complex SVs for altering phenotypes.

RtsA and RtsB Coordinately Regulate Expression of the Invasion and Flagellar Genes in Salmonella enterica Serovar Typhimurium

ABSTRACT Salmonella enterica serovar Typhimurium encounters numerous host environments and defense mechanisms during the infection process. The bacterium responds by tightly regulating the expression of virulence genes. We identified two regulatory proteins, termed RtsA and RtsB, which are encoded in an operon located on an island integrated at tRNAPheU in S. enterica serovar Typhimurium. RtsA belongs to the AraC/XylS family of regulators, and RtsB is a helix-turn-helix DNA binding protein. In a random screen, we identified five RtsA-regulated fusions, all belonging to the Salmonella pathogenicity island 1 (SPI1) regulon, which encodes a type III secretion system (TTSS) required for invasion of epithelial cells. We show that RtsA increases expression of the invasion genes by inducing hilA expression. RtsA also induces expression of hilD, hilC, and the invF operon. However, induction of hilA is independent of HilC and HilD and is mediated by direct binding of RtsA to the hilA promoter. The phenotype of an rtsA null mutation is similar to the phenotype of a hilC mutation, both of which decrease expression of SPI1 genes approximately twofold. We also show that RtsA can induce expression of a SPI1 TTSS effector, slrP, independent of any SPI1 regulatory protein. RtsB represses expression of the flagellar genes by binding to the flhDC promoter region. Repression of the positive activators flhDC decreases expression of the entire flagellar regulon. We propose that RtsA and RtsB coordinate induction of invasion and repression of motility in the small intestine.

Interactions between two fission yeast serine/arginine-rich proteins and their modulation by phosphorylation

The unexpected low number of genes in the human genome has triggered increasing attention to alternative pre-mRNA splicing, and serine/arginine-rich (SR) proteins have been correlated with the complex alternative splicing that is a characteristic of metazoans. SR proteins interact with RNA and splicing protein factors, and they also undergo reversible phosphorylation, thereby regulating constitutive and alternative splicing in mammals and Drosophila. However, it is not clear whether the features of SR proteins and alternative splicing are present in simple and genetically tractable organisms, such as yeasts. In the present study, we show that the SR-like proteins Srp1 and Srp2, found in the fission yeast Schizosaccharomyces pombe, interact with each other and the interaction is modulated by protein phosphorylation. By using Srp1 as bait in a yeast two-hybrid analysis, we specifically isolated Srp2 from a random screen. This Srp interaction was confirmed by a glutathione-S-transferase pull-down assay. We also found that the Srp1—Srp2 complex was phosphorylated at a reduced efficiency by a fission yeast SR-specific kinase, Dis1-suppression kinase (Dsk1). Conversely, Dsk1-mediated phosphorylation inhibited the formation of the Srp complex. These findings offer the first example in fission yeast for interactions between SR-related proteins and the modulation of the interactions by specific protein phosphorylation, suggesting that a mammalian-like SR protein function may exist in fission yeast.