CRISPR-Cas systems provide bacteria with adaptive immunity against bacteriophages 1 . However, DNA modification 2,3 , the production of anti-CRISPR proteins 4,5 and potentially other strategies enable phages to evade CRISPR-Cas. Here we discovered a Serratia jumbophage that evaded type I CRISPR-Cas systems, but was sensitive to type III immunity. Jumbophage infection resulted in a nucleus-like structure enclosed by a proteinaceous phage shella phenomenon only reported recently for distantly related Pseudomonas phages 6,7 . All three native CRISPR-Cas complexes in Serratia -type I-E, I-F and III-A -were spatially excluded from the phage nucleus and phage DNA was not targeted. However, the type III-A system still arrested jumbophage infection by targeting phage RNA in the cytoplasm in a process requiring Cas7, Cas10 and an accessory nuclease. Type III, but not type I, systems frequently targeted nucleus-forming jumbophages that were identified in global viral sequence datasets. These findings explain why many bacteria harbour both RNA-and DNA-targeting CRISPR-Cas systems 1,8 . Together, our results indicate that jumbophage nucleus-like compartments serve as a barrier to DNA-targeting, but not RNA-targeting defences, and that this phenomenon is widespread amongst jumbophages.

No abstract

In this work, we studied the role of surface layer (S-layer) proteins in the adaptation of Lactobacillus acidophilus ATCC 4356 to the osmotic stress generated by high salt. The amounts of the predominant and the auxiliary S-layer proteins SlpA and SlpX were strongly influenced by the growth phase and high-salt conditions (0.6 M NaCl). Changes in gene expression were also observed as the mRNAs of the slpA and slpX genes increased related to the growth phase and presence of high salt. A growth stage-dependent modification on the S-layer protein profile in response to NaCl was observed: while in control conditions, the auxiliary SlpX protein represented less than 10 % of the total S-layer protein, in high-salt conditions, it increased to almost 40 % in the stationary phase. The increase in S-layer protein synthesis in the stress condition could be a consequence of or a way to counteract the fragility of the cell wall, since a decrease in the cell wall thickness and envelope components (peptidoglycan layer and lipoteichoic acid content) was observed in L. acidophilus when compared to a non-S-layer-producing species such as Lactobacillus casei. Also, the stationary phase and growth in high-salt medium resulted in increased release of S-layer proteins to the supernatant medium. Overall, these findings suggest that pre-growth in high-salt conditions would result in an advantage for the probiotic nature of L. acidophilus ATCC 4356 as the increased amount and release of the S-layer might be appropriate for its antimicrobial capacity.

No abstract

Graphical AbstractHighlights d Imprecisely acquired (slipped) spacers are impaired for CRISPR-Cas immunity d À1 and +1 slipped spacers stimulate primed CRISPR adaptation d Slipped spacers enhance priming before phage escape mutations arise d Slipping enhances CRISPR-Cas resilience to phage mutation by increasing immune diversity SUMMARY Many prokaryotes possess CRISPR-Cas adaptive immune systems to defend against viruses and invading mobile genetic elements. CRISPR-Cas immunity relies on genetic memories, termed spacers, for sequence-specific recognition of infections. The diversity of spacers within host populations is important for immune resilience, but we have limited understanding of how CRISPR diversity is generated. Type I CRISPR-Cas systems use existing spacers to enhance the acquisition of new spacers through primed CRISPR adaptation (priming). Here, we present a pathway to priming that is stimulated by imprecisely acquired (slipped) spacers. Slipped spacers are less effective for immunity but increase priming compared with canonical spacers. The benefits of slipping depend on the relative rates of phage mutation and adaptation during defense. We propose that slipped spacers provide a route to increase population-level spacer diversity that pre-empts phage escape mutant proliferation and that the trade-off between adaptation and immunity is important in diverse CRISPR-Cas systems.