ABSTRACT
Bacteria have evolved sophisticated adaptive immune systems, called CRISPR-Cas, that provide sequence-specific protection against phage infection. In turn, phages have evolved a broad spectrum of anti-CRISPRs that suppress these immune systems. Here we report structures of anti-CRISPR protein IF9 (AcrIF9) in complex with the type I-F CRISPR RNA-guided surveillance complex (Csy). In addition to sterically blocking the hybridization of complementary dsDNA to the CRISPR RNA, our results show that AcrIF9 binding also promotes non-sequence-specific engagement with dsDNA, potentially sequestering the complex from target DNA. These findings highlight the versatility of anti-CRISPR mechanisms utilized by phages to suppress CRISPR-mediated immune systems.
Subject(s)
Bacteria/metabolism , Bacteriophages/metabolism , CRISPR-Cas Systems , DNA/metabolism , RNA, Guide, Kinetoplastida/metabolism , Viral Proteins/metabolism , Amino Acid Sequence , Bacteria/genetics , Bacteria/virology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacteriophages/genetics , Cryoelectron Microscopy , DNA/chemistry , DNA/genetics , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , Nucleic Acid Conformation , Protein Binding , Protein Conformation , Proteus penneri/genetics , Proteus penneri/metabolism , Proteus penneri/virology , RNA, Guide, Kinetoplastida/chemistry , RNA, Guide, Kinetoplastida/genetics , Sequence Homology, Amino Acid , Viral Proteins/chemistry , Viral Proteins/geneticsABSTRACT
INTRODUCTION: The aim of this study was to evaluate betalactam resistance within the genus Proteus and characterize the betalactamases responsible for this resistance. METHODS: We analyzed 99 strains (87, P. mirabilis; 10 P. vulgaris, and 2, P. penneri) isolated from patients at one University Hospital. Antibiotic susceptibility tests were performed according to NCCLS recommendations. Presence of extended spectrum betalactamases (ESBL) was inferred by both double disk diffusion tests and minimum inhibitory concentration (MIC) of third and fourth generation cephalosporins alone and in the presence of clavulanic acid. Isoelectric points (pI) of the enzymes were estimated by isoelectrofocusing and the presence of the encoding genes was confirmed by polymerase chain reaction (PCR). RESULTS: A broad spectrum betalactamase could be detected in those isolates (28%) resistant to penicillin and first generation cephalosporins while CTX-M-2 enzyme could be detected in P. mirabilis isolates resistant to third and fourth generation cephalosporins (18%). One of the P. vulgaris displayed reduced susceptibility to cefotaxime due to an enzyme of pI 7.4, while resistance to cefotaxime in one P. penneri was related to an enzyme of pI 6.8. Both enzymes were active on cefotaxime (1,000 mg/l) in the iodometric assay. CONCLUSION: The broad extended spectrum betalactamase within genus Proteus was TEM-1, while CTX-M-2 was the ESBL responsible for the third and fourth generation cephalosporins in P. mirabilis. In P. vulgaris and P. penneri this resistance was associated with the hyperproduction of the chromosomal encoded betalactamase.
