CRISPR-Cas is a bacterial and archaeal adaptive immune system that uses short, invader-derived sequences termed spacers to target invasive nucleic acids. Upon recognition of previously encountered invaders, the system can stimulate secondary spacer acquisitions, a process known as primed adaptation. Previous studies of primed adaptation have been complicated by intrinsically high interference efficiency of most systems against bona fide targets. As such, most primed adaptation to date has been studied within the context of imperfect sequence complementarity between spacers and targets. Here, we take advantage of a native type I-C CRISPR-Cas system in that displays robust primed adaptation even within the context of a perfectly matched target. Using next-generation sequencing to survey acquired spacers, we observe strand bias and positional preference that are consistent with a 3'-5' translocation of the adaptation machinery. We show that spacer acquisition happens in a wide range of frequencies across the plasmid, including a remarkable hotspot that predominates irrespective of the priming strand. We systematically characterize protospacer sequence constraints in both adaptation and interference and reveal extensive flexibilities regarding the protospacer adjacent motif in both processes. Lastly, in a strain with a genetically truncated CRISPR array, we observe increased interference efficiency, which, when coupled with forced maintenance of a targeted plasmid, provides a useful experimental system to study spacer loss. Based on these observations, we propose that the type I-C system represents a powerful model to study primed adaptation and the interplay between CRISPR interference and adaptation.

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16CRISPR-Cas is a bacterial and archaeal adaptive immune system that uses short, invader-derived 17 sequences termed spacers to target invasive nucleic acids. Upon recognition of previously encountered 18 invaders, the system can stimulate secondary spacer acquisitions, a process known as primed adaptation. 19Previous studies of primed adaptation have been complicated by intrinsically high interference efficiency 20 of most systems against bona fide targets. As such, most primed adaptation to date has been studied 21 within the context of imperfect sequence complementarity between spacers and targets. Here, we take 22 advantage of a native type I-C CRISPR-Cas system in Legionella pneumophila that displays robust 23 primed adaptation even within the context of a perfectly matched target. Using next-generation 24 sequencing to survey acquired spacers, we observe strand bias and positional preference that are 25 consistent with a 3′ to 5′ translocation of the adaptation machinery. We show that spacer acquisition 26 happens in a wide range of frequencies across the plasmid, including a remarkable hotspot that 27 predominates irrespective of the priming strand. We systematically characterize protospacer sequence 28 constraints in both adaptation and interference and reveal extensive flexibilities regarding the protospacer 29 adjacent motif in both processes. Lastly, in a strain with a genetically truncated CRISPR array, we 30 observe greatly increased interference efficiency coupled with a dramatic shift away from spacer 31 acquisition towards spacer loss. Based on these observations, we propose that the Legionella type I-C 32 system represents a powerful model to study primed adaptation and the interplay between CRISPR 33 interference and adaptation.

Coagulase-negative staphylococci and Staphylococcus aureus colonize similar niches in mammals and conceivably compete for space and nutrients. Here, we report that a coagulase-negative staphylococcus, Staphylococcus chromogenes ATCC43764, synthesizes and secretes 6-thioguanine (6-TG), a purine analog that suppresses S. aureus growth by inhibiting de novo purine biosynthesis. We identify a 6-TG biosynthetic gene cluster in S. chromogenes and other coagulase-negative staphylococci including S. epidermidis, S. pseudintermedius and S. capitis. Recombinant S. aureus strains harbouring this operon produce 6-TG and, when used in subcutaneous co-infections in mice with virulent S. aureus USA300, protect the host from necrotic lesion formation. Used prophylactically, 6-TG reduces necrotic skin lesions in mice infected with USA300, and this effect is mediated by abrogation of toxin production. RNAseq analyses reveal that 6-TG downregulates expression of genes coding for purine biosynthesis, the accessory gene regulator (agr) and ribosomal proteins in S. aureus, providing an explanation for its effect on toxin production.

Energy-coupling factor type transporters (ECF) represent trace nutrient acquisition systems. Substrate binding components of ECF-transporters are membrane proteins with extraordinary affinity, allowing them to scavenge trace amounts of ligand. A number of molecules have been described as substrates of ECF-transporters, but an involvement in iron-acquisition is unknown. Host-induced iron limitation during infection represents an effective mechanism to limit bacterial proliferation. We identified the iron-regulated ECF-transporter Lha in the opportunistic bacterial pathogen Staphylococcus lugdunensis and show that the transporter is specific for heme. The recombinant substrate-specific subunit LhaS accepted heme from diverse host-derived hemoproteins. Using isogenic mutants and recombinant expression of Lha, we demonstrate that its function is independent of the canonical heme acquisition system Isd and allows proliferation on human cells as sources of nutrient iron. Our findings reveal a unique strategy of nutritional heme acquisition and provide the first example of an ECF-transporter involved in overcoming host-induced nutritional limitation.

We recently discovered that 6-thioguanine (6-TG) is an anti-virulence compound that is produced by a number of coagulase negative staphylococci. In Staphylococcus aureus , it inhibits de novo purine biosynthesis and ribosomal protein expression, thus inhibiting growth and abrogating toxin production. Mechanisms by which S. aureus may develop resistance to this compound are currently unknown. Here, we show that 6-TG-resistant S. aureus mutants emerge spontaneously when the bacteria are subjected to high concentrations of 6-TG in vitro . Whole genome sequencing of these mutants revealed frameshift and missense mutations in a xanthine-uracil permease family protein ( stgP : s ix t hio g uanine p ermease) and single nucleotide polymorphisms in hypoxanthine phosphoribosyltransferase ( hpt ). These mutations engender S. aureus the ability to resist both the growth inhibitory and toxin down regulation effects of 6-TG. While prophylactic administration of 6-TG ameliorates necrotic lesions in subcutaneous infection of mice with MRSA strain USA300-LAC, the drug did not reduce lesion size formed by the 6-TG resistant strains. These findings identify mechanisms of 6-TG resistance and this information can be leveraged to inform strategies to slow the evolution of resistance.

Coagulase-negative staphylococci (CoNS) are frequently commensal bacteria that rarely cause disease in mammals. Staphylococcus lugdunensis is an exceptional CoNS that causes disease in humans similar to virulent Staphylococcus aureus , but the factors that enhance the virulence of this bacterium remain ill defined. Here, we used random transposon insertion mutagenesis to identify the agr quorum sensing system as a regulator of hemolysins in S. lugdunensis .

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