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.

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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.

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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.

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Bacterial pathogens are major causes of crop diseases, leading to significant production losses. For instance, kiwifruit canker, caused by the phytopathogen Pseudomonas syringae pv. actinidiae (Psa), has posed a global challenge to kiwifruit production. Treatment with copper and antibiotics, whilst initially effective, is leading to the rise of bacterial resistance, requiring new biocontrol approaches. Previously, we isolated a group of closely related Psa phages with biocontrol potential, which represent environmentally sustainable antimicrobials. However, their deployment as antimicrobials requires further insight into their properties and infection strategy. Here, we provide an in‐depth examination of the genome of ΦPsa374‐like phages and show that they use lipopolysaccharides (LPS) as their main receptor. Through proteomics and cryo‐electron microscopy of ΦPsa374, we revealed the structural proteome and that this phage possess a T = 9 capsid triangulation, unusual for myoviruses. Furthermore, we show that ΦPsa374 phage resistance arises in planta through mutations in a glycosyltransferase involved in LPS synthesis. Lastly, through in vitro evolution experiments we showed that phage resistance is overcome by mutations in a tail fibre and structural protein of unknown function in ΦPsa374. This study provides new insight into the properties of ΦPsa374‐like phages that informs their use as antimicrobials against Psa.

During infection, phages manipulate bacteria to redirect metabolism towards viral proliferation. To counteract phages, some bacteria employ CRISPR-Cas systems that provide adaptive immunity. While CRISPR-Cas mechanisms have been studied extensively, their effects on both the phage and the host during phage infection remains poorly understood. Here, we analysed the infection of Serratia by a siphovirus (JS26) and the transcriptomic response with, or without type I-E or I-F CRISPR-Cas immunity. In non-immune Serratia, phage infection altered bacterial metabolism by upregulating anaerobic respiration and amino acid biosynthesis genes, while flagella production was suppressed. Furthermore, phage proliferation required a late-expressed viral Cas4 homologue, which did not influence CRISPR adaptation. While type I-E and I-F immunity provided robust defence against phage infection, phage development still impacted the bacterial host. Moreover, DNA repair and SOS response pathways were upregulated during type I immunity. We also discovered that the type I-F system is controlled by a positive autoregulatory feedback loop that is activated upon phage targeting during type I-F immunity, leading to a controlled anti-phage response. Overall, our results provide new insight into phage-host dynamics and the impact of CRISPR immunity within the infected cell.