Prokaryotes encode various host defense systems that provide protection against mobile genetic elements. Restriction–modification (R–M) and CRISPR–Cas systems mediate host defense by sequence specific targeting of invasive DNA. T-even bacteriophages employ covalent modifications of nucleobases to avoid binding and therefore cleavage of their DNA by restriction endonucleases. Here, we describe that DNA glucosylation of bacteriophage genomes affects interference of some but not all CRISPR–Cas systems. We show that glucosyl modification of 5-hydroxymethylated cytosines in the DNA of bacteriophage T4 interferes with type I-E and type II-A CRISPR–Cas systems by lowering the affinity of the Cascade and Cas9–crRNA complexes for their target DNA. On the contrary, the type V-A nuclease Cas12a (also known as Cpf1) is not impaired in binding and cleavage of glucosylated target DNA, likely due to a more open structural architecture of the protein. Our results suggest that CRISPR–Cas systems have contributed to the selective pressure on phages to develop more generic solutions to escape sequence specific host defense systems.

While many adhesion receptors are known to influence tumor progression, the mechanisms by which they dynamically regulate cell-cell adhesion remain elusive. We previously identified Activated Leukocyte Cell Adhesion Molecule (ALCAM) as a clinically relevant driver of metastasis and hypothesized that a tunable mechanism of ectodomain shedding regulates its contribution to dissemination. To test this hypothesis, we examined an under-explored ALCAM splice variant (ALCAM-Iso2) and demonstrated that loss of the membrane-proximal region of ALCAM (exon 13) increased metastasis four-fold. Mechanistic studies identified a novel MMP14-dependent membrane distal cleavage site in ALCAM-Iso2, which mediated a ten-fold increase in shedding, thereby decreasing cellular cohesion. Importantly, the loss of cohesion is not limited to the cell capable of shedding because the released extracellular domain diminished cohesion of non-shedding cells through disruption of ALCAM-ALCAM interactions. ALCAM-Iso2-dominated expression in bladder cancer tissue, compared to normal bladder, further emphasizes that ALCAM alternative splicing may contribute to clinical disease progression. The requirement for both the loss of exon 13 and the gain of metalloprotease activity suggests that ALCAM shedding and concomitant regulation of tumor cell adhesion is a locally tunable process.

Towards a synthetic cell: designing, testing and optimizing functional genomic modules in E. coli

Prokaryotic genes encoding functionally related proteins are often clustered in operons. The compact structure of operons allows for co-transcription of the genes, and for co-translation of the polycistronic messenger RNA to the corresponding proteins. This leads to reduced regulatory complexity and enhanced gene expression efficiency, and as such to an overall metabolic benefit for the protein production process in bacteria and archaea. Interestingly, the genes encoding the subunits of one of the most conserved and ubiquitous protein complexes, the RNA polymerase, are not clustered in a single operon. Rather, its genes are scattered in all known prokaryotic genomes, generally integrated in different ribosomal operons. To analyze the impact of this genetic organization on the fitness of Escherichia coli, we constructed a bacterial artificial chromosome harboring the genes encoding the RNA polymerase complex in a single operon. Subsequent deletion of the native chromosomal genes led to a reduced growth on minimal medium. However, by using adaptive laboratory evolution the growth rate was restored to wild-type level. Hence, we show that a highly conserved genetic organization of core genes in a bacterium can be reorganized by a combination of design, construction and optimization, yielding a well-functioning synthetic genetic architecture.