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.

Silica has been frequently studied using infrared and Raman spectroscopy due to its importance in many practical contexts where its surface chemistry plays a vital role. The majority of these studies have utilized chemical-vapor-deposited films in vacuo after high-temperature calcination. However, room-temperature hydration and dehydration of thin silica particle films has not been well characterized in spite of the importance of such films as substrates for polymer and surfactant adsorption. The present study has utilized ATR-IR spectroscopy and thin silica particle films exposed to varying humidity to clearly show reversible conversion between surface siloxanes and hydrogen-bonded silanols without the need for semiempirical peak deconvolution. The IR spectra from corresponding hydration experiments on deuterated silica films has confirmed the vibrational mode assignments. The variation of humidity over silica films formed from silica suspensions of differing pH gave IR spectra consistent with the change in the relative populations of siloxide to silanol surface groups. In addition, total internal reflection Raman spectroscopy has been used to provide further evidence of room-temperature dehydroxylation, with spectral evidence for the presence of three-membered siloxane rings when films are dehydrated under argon. The confirmation of room-temperature siloxane-to-silanol interconversion is expected to benefit understanding in many silica surface chemical contexts.

The common polysaccharide antigen (CPA) of the lipopolysaccharide (LPS) from Pseudomonas syringae is highly variable, but the genetic basis for this is poorly understood. We have characterized the CPA locus from P. syringae pv. actinidiae (Psa). This locus has genes for Land D-rhamnose biosynthesis and an operon coding for ABC transporter subunits, a bifunctional glycosyltransferase and an O-methyltransferase. This operon is predicted to have a role in the transport, elongation and termination of the CPA oligosaccharide and is referred to as the TET operon. Two alleles of the TET operon were present in different biovars (BV) of Psa and lineages of the closely related pathovar P. syringae pv. actinidifoliorum. This allelic variation was reflected in the electrophoretic properties of purified LPS from the different isolates. Gene knockout of the TET operon allele from BV1 and replacement with that from BV3, demonstrated the link between the genetic locus and the biochemical properties of the LPS molecules in Psa. Sequence analysis of the TET operon from a range of P. syringae and P. viridiflava isolates displayed a phylogenetic history incongruent with core gene phylogeny but correlates with previously reported tailocin sensitivity, suggesting a functional relationship between LPS structure and tailocin susceptibility.

No abstract

Bacteriophage ϕPsa21 is a potential biocontrol agent that infects the kiwifruit phytopathogen Pseudomonas syringae pv. actinidiae. ϕPsa21 is a “jumbo” phage with a genome of ∼305 kb.

Identifying the ligands sensed by chemoreceptors remains challenging, in part because current screening methods are low-throughput, costly, and/or time-consuming. In contrast, fluorescence thermal shift (FTS) assays provide a fast and inexpensive approach to chemoreceptor-ligand screening. In FTS assays, the temperature at which a protein denatures is measured by monitoring the fluorescence of a dye with affinity for hydrophobic regions of the protein, which are exposed as the protein unfolds. A detectable increase (or "shift") in the melting temperature (T ) of the protein in the presence of a potential ligand indicates binding. Here, we present our protocol for using FTS assays for the screening of chemoreceptor ligands in a high-throughput, 96-well plate format. We have also included details on the use of Biolog Phenotype Microarray plates as a convenient ligand library, although the methods described should be generally applicable to other library formats as well.

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.

The rise of antibiotic resistance, coupled with increased expectations for mobility in later life, is creating a need for biofilm inhibitors and delivery systems that will reduce surgical implant infection. A limitation of some of these existing delivery approaches is toxicity exhibited toward host cells. Here, we report the application of a novel inhibitor of the enzyme, methylthioadenosine nucleosidase (MTAN), a key enzyme in bacterial metabolic pathways, which include S-adenosylmethionine catabolism and purine nucleotide recycling, in combination with a poly­(vinyl alcohol)-tyramine-based (PVA-Tyr) hydrogel delivery system. We demonstrate that a lead MTAN inhibitor, selected from a screened library of 34 candidates, (2S)-2-(4-amino-5H-pyrrolo3,2-dpyrimidin-7-ylmethyl)­aminoundecan-1-ol (31), showed a minimum biofilm inhibitory concentration of 2.2 ± 0.4 μM against a clinical staphylococcal species isolated from an infected implant. We observed that extracellular DNA, a key constituent of biofilms, is significantly reduced when treated with 10 μM compound 31, along with a decrease in biofilm thickness. Compound 31 was incorporated into a hydrolytically degradable photo-cross-linked PVA-Tyr hydrogel and the release profile was evaluated by HPLC studies. Compound 31 released from the PVA-hydrogel system significantly reduced biofilm formation (77.2 ± 8.4% biofilm inhibition). Finally, compound 31 released from PVA-Tyr showed no negative impact on human bone marrow stromal cell (MSC) viability, proliferation, or morphology. The results demonstrate the potential utility of MTAN inhibitors in treating infections caused by Gram-positive bacteria, and the development of a nontoxic release system that has potential for tunability for time scale of delivery.