Discovery of a rapidly evolving yeast defense factor, KTD1, against the secreted killer toxin K28.

Secreted protein toxins are widely used weapons in conflicts between organisms. Elucidating how organisms genetically adapt to defend themselves against these toxins is fundamental to understanding the coevolutionary dynamics of competing organisms. Within yeast communities, "killer" toxins are secreted to kill nearby sensitive yeast, providing a fitness advantage in competitive growth environments. Natural yeast isolates vary in their sensitivity to these toxins, but to date, no polymorphic genetic factors contributing to defense have been identified. We investigated the variation in resistance to the killer toxin K28 across diverse natural isolates of the Saccharomyces cerevisiae population. Using large-scale linkage mapping, we discovered a novel defense factor, which we named KTD1. We identified many KTD1 alleles, which provided different levels of K28 resistance. KTD1 is a member of the DUP240 gene family of unknown function, which is rapidly evolving in a region spanning its two encoded transmembrane helices. We found that this domain is critical to KTD1's protective ability. Our findings implicate KTD1 as a key polymorphic factor in the defense against K28 toxin.

Systematic Dissection of Sequence Elements Controlling σ70 Promoters Using a Genomically Encoded Multiplexed Reporter Assay in Escherichia coli.

Promoters are the key drivers of gene expression and are largely responsible for the regulation of cellular responses to time and environment. In Escherichia coli, decades of studies have revealed most, if not all, of the sequence elements necessary to encode promoter function. Despite our knowledge of these motifs, it is still not possible to predict the strength and regulation of a promoter from primary sequence alone. Here we develop a novel multiplexed assay to study promoter function in E. coli by building a site-specific genomic recombination-mediated cassette exchange system that allows for the facile construction and testing of large libraries of genetic designs integrated into precise genomic locations. We build and test a library of 10898 σ70 promoter variants consisting of all combinations of a set of eight -35 elements, eight -10 elements, three UP elements, eight spacers, and eight backgrounds. We find that the -35 and -10 sequence elements can explain approximately 74% of the variance in promoter strength within our data set using a simple log-linear statistical model. Simple neural network models explain >95% of the variance in our data set by capturing nonlinear interactions with the spacer, background, and UP elements.

SARS-CoV-2 omicron BA.2.87.1 exhibits higher susceptibility to serum neutralization than EG.5.1 and JN.1.

As SARS-CoV-2 continues to spread and mutate, tracking the viral evolutionary trajectory and understanding the functional consequences of its mutations remain crucial. Here, we characterized the antibody evasion, ACE2 receptor engagement, and viral infectivity of the highly mutated SARS-CoV-2 Omicron subvariant BA.2.87.1. Compared with other Omicron subvariants, including EG.5.1 and the current predominant JN.1, BA.2.87.1 exhibits less immune evasion, reduced viral receptor engagement, and comparable infectivity in Calu-3 lung cells. Intriguingly, two large deletions (Δ15-26 and Δ136-146) in the N-terminal domain (NTD) of the spike protein facilitate subtly increased antibody evasion but significantly diminish viral infectivity. Collectively, our data support the announcement by the USA CDC that the public health risk posed by BA.2.87.1 appears to be low.

Stool protein mass spectrometry identifies biomarkers for the early detection of diffuse-type gastric cancer.

There is a high unmet need for early detection approaches for diffuse gastric cancer (DGC). We examined whether the stool proteome of mouse models of GC or individuals with hereditary diffuse GC (HDGC) have utility as biomarkers for early detection. Proteomic mass spectrometry of stool from a genetically engineered mouse model driven by oncogenic KrasG12D and loss of p53 and Cdh1 in gastric parietal cells (known as TCON mice) identified differentially abundant proteins compared to littermate controls. Immunoblot assays validated a panel of proteins including actinin alpha 4 (ACTN4), N-acylsphingosine amidohydrolase 2 (ASAH2), dipeptidyl peptidase 4 (DPP4), and valosin-containing protein (VCP) as enriched in TCON stool compared to littermate control stool. Immunofluorescence analysis of these proteins in TCON stomach sections revealed increased protein expression as compared to littermate controls. Proteomic mass spectrometry of stool obtained from HDGC patients with CDH1 mutations identified increased expression of ASAH2, DPP4, VCP, lactotransferrin (LTF), and tropomyosin-2 (TPM2) relative to stool from healthy sex and age-matched donors. Chemical inhibition of ASAH2 using C6-urea ceramide was toxic to GC cell lines and patient derived-GC organoids. This toxicity was reversed by adding downstream products of the S1P synthesis pathway, suggesting a dependency on ASAH2 activity in GC. An exploratory analysis of the HDGC stool microbiome identified features which correlated with patient tumors. Here we provide evidence supporting the potential of analyzing stool biomarkers for the early detection of DGC.

Multiplexed characterization of rationally designed promoter architectures deconstructs combinatorial logic for IPTG-inducible systems.

A crucial step towards engineering biological systems is the ability to precisely tune the genetic response to environmental stimuli. In the case of Escherichia coli inducible promoters, our incomplete understanding of the relationship between sequence composition and gene expression hinders our ability to predictably control transcriptional responses. Here, we profile the expression dynamics of 8269 rationally designed, IPTG-inducible promoters that collectively explore the individual and combinatorial effects of RNA polymerase and LacI repressor binding site strengths. We then fit a statistical mechanics model to measured expression that accurately models gene expression and reveals properties of theoretically optimal inducible promoters. Furthermore, we characterize three alternative promoter architectures and show that repositioning binding sites within promoters influences the types of combinatorial effects observed between promoter elements. In total, this approach enables us to deconstruct relationships between inducible promoter elements and discover practical insights for engineering inducible promoters with desirable characteristics.

Discovering the Molecular Determinants of Phaeobacter inhibens Susceptibility to Phaeobacter Phage MD18.

Bacteriophages have immense potential as antibiotic therapies and in genetic engineering. Understanding the mechanisms that bacteriophages implement to infect their hosts will allow researchers to manipulate these systems and adapt them to specific bacterial targets. In this study, we isolated a bacteriophage capable of infecting the marine alphaproteobacterium Phaeobacter inhibens and determined its mechanism of infection. Phaeobacter virus MD18, a novel species of bacteriophage isolated in Woods Hole, MA, exhibits potent lytic ability against P. inhibens and appears to be of the Siphoviridae morphotype. The genomic sequence of MD18 displayed significant similarity to another siphophage, the recently discovered Roseobacter phage DSS3P8, but genomic and phylogenetic analyses, assessing host range and a search of available metagenomes are all consistent with the conclusion that Phaeobacter phage MD18 is a novel lytic phage. We incubated MD18 with a library of barcoded P. inhibens transposon insertion mutants and identified 22 genes that appear to be required for phage predation of this host. Network analysis of these genes using genomic position, Gene Ontology (GO) term enrichment, and protein associations revealed that these genes are enriched for roles in assembly of a type IV pilus (T4P) and regulators of cellular morphology. Our results suggest that T4P serve as receptors for a novel marine virus that targets P. inhibens.IMPORTANCE Bacteriophages are useful nonantibiotic therapeutics for bacterial infections as well as threats to industries utilizing bacterial agents. This study identified Phaeobacter virus MD18, a phage antagonist of Phaeobacter inhibens, a bacterium with promising use as a probiotic for aquatic farming industries. Genomic analysis suggested that Phaeobacter phage MD18 has evolved to enhance its replication in P. inhibens by adopting favorable tRNA genes as well as through genomic sequence adaptation to resemble host codon usage. Lastly, a high-throughput analysis of P. inhibens transposon insertion mutants identified genes that modulate host susceptibility to phage MD18 and implicated the type IV pilus as the likely receptor recognized for adsorption. This study marks the first characterization of the relationship between P. inhibens and an environmentally sampled phage, which informs our understanding of natural threats to the bacterium and may promote the development of novel phage technologies for genetic manipulation of this host.

A haploid genetics toolbox for Arabidopsis thaliana.

Genetic analysis in haploids provides unconventional yet powerful advantages not available in diploid organisms. In Arabidopsis thaliana, haploids can be generated through seeds by crossing a wild-type strain to a transgenic strain with altered centromeres. Here we report the development of an improved haploid inducer (HI) strain, SeedGFP-HI, that aids selection of haploid seeds prior to germination. We also show that haploids can be used as a tool to accelerate a variety of genetic analyses, specifically pyramiding multiple mutant combinations, forward mutagenesis screens, scaling down a tetraploid to lower ploidy levels and swapping of nuclear and cytoplasmic genomes. Furthermore, the A. thaliana HI can be used to produce haploids from a related species A. suecica and generate homozygous mutant plants from strong maternal gametophyte lethal alleles, which is not possible via conventional diploid genetics. Taken together, our results demonstrate the utility and power of haploid genetics in A. thaliana.