Resistance mutations generate divergent antibiotic susceptibility profiles against translation inhibitors.

Mutations conferring resistance to translation inhibitors often alter the structure of rRNA. Reduced susceptibility to distinct structural antibiotic classes may, therefore, emerge when a common ribosomal binding site is perturbed, which significantly reduces the clinical utility of these agents. The translation inhibitors negamycin and tetracycline interfere with tRNA binding to the aminoacyl-tRNA site on the small 30S ribosomal subunit. However, two negamycin resistance mutations display unexpected differential antibiotic susceptibility profiles. Mutant U1060A in 16S Escherichia coli rRNA is resistant to both antibiotics, whereas mutant U1052G is simultaneously resistant to negamycin and hypersusceptible to tetracycline. Using a combination of microbiological, biochemical, single-molecule fluorescence transfer experiments, and X-ray crystallography, we define the specific structural defects in the U1052G mutant 70S E. coli ribosome that explain its divergent negamycin and tetracycline susceptibility profiles. Unexpectedly, the U1052G mutant ribosome possesses a second tetracycline binding site that correlates with its hypersusceptibility. The creation of a previously unidentified antibiotic binding site raises the prospect of identifying similar phenomena in antibiotic-resistant pathogens in the future.

Staphylococcus epidermidis Csm1 is a 3'-5' exonuclease.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) offer an adaptive immune system that protects bacteria and archaea from nucleic acid invaders through an RNA-mediated nucleic acid cleavage mechanism. Our knowledge of nucleic acid cleavage mechanisms is limited to three examples of widely different ribonucleoprotein particles that target either DNA or RNA. Staphylococcus epidermidis belongs to the Type III-A CRISPR system and has been shown to interfere with invading DNA in vivo. The Type III-A CRISPR system is characterized by the presence of Csm1, a member of Cas10 family of proteins, that has a permuted histidine-aspartate domain and a nucleotidyl cyclase-like domain, both of which contain sequence features characteristic of nucleases. In this work, we show in vitro that a recombinant S. epidermidis Csm1 cleaves single-stranded DNA and RNA exonucleolytically in the 3'-5' direction. We further showed that both cleavage activities are divalent-metal-dependent and reside in the GGDD motif of the cyclase-like domain. Our data suggest that Csm1 may work in the context of an effector complex to degrade invading DNA and participate in CRISPR RNA maturation.

The RNA-binding domain of bacteriophage P22 N protein is highly mutable, and a single mutation relaxes specificity toward lambda.

Antitermination in bacteriophage P22, a lambdoid phage, uses the arginine-rich domain of the N protein to recognize boxB RNAs in the nut site of two regulated transcripts. Using an antitermination reporter system, we screened libraries in which each nonconserved residue in the RNA-binding domain of P22 N was randomized. Mutants were assayed for the ability to complement N-deficient virus and for antitermination with P22 boxB(left) and boxB(right) reporters. Single amino acid substitutions complementing P22 N(-) virus were found at 12 of the 13 positions examined. We found evidence for defined structural roles for seven nonconserved residues, which was generally compatible with the nuclear magnetic resonance model. Interestingly, a histidine can be replaced by any other aromatic residue, although no planar partner is obvious. Few single substitutions showed bias between boxB(left) and boxB(right), suggesting that the two RNAs impose similar constraints on genetic drift. A separate library comprising only hybrids of the RNA-binding domains of P22, lambda, and phi21 N proteins produced mutants that displayed bias. P22 N(-) plaque size plotted against boxB(left) and boxB(right) reporter activities suggests that lytic viral fitness depends on balanced antitermination. A few N proteins were able to complement both lambda N- and P22 N-deficient viruses, but no proteins were found to complement both P22 N- and phi21 N-deficient viruses. A single tryptophan substitution allowed P22 N to complement both P22 and lambda N(-). The existence of relaxed-specificity mutants suggests that conformational plasticity provides evolutionary transitions between distinct modes of RNA-protein recognition.

Bacteriophage P22 antitermination boxB sequence requirements are complex and overlap with those of lambda.

Transcription antitermination in phages lambda and P22 uses N proteins that bind to similar boxB RNA hairpins in regulated transcripts. In contrast to the lambda N-boxB interaction, the P22 N-boxB interaction has not been extensively studied. A nuclear magnetic resonance structure of the P22 N peptide boxB(left) complex and limited mutagenesis have been reported but do not reveal a consensus sequence for boxB. We have used a plasmid-based antitermination system to screen boxBs with random loops and to test boxB mutants. We find that P22 N requires boxB to have a GNRA-like loop with no simple requirements on the remaining sequences in the loop or stem. U:A or A:U base pairs are strongly preferred adjacent to the loop and appear to modulate N binding in cooperation with the loop and distal stem. A few GNRA-like hexaloops have moderate activity. Some boxB mutants bind P22 and lambda N, indicating that the requirements imposed on boxB by P22 N overlap those imposed by lambda N. Point mutations can dramatically alter boxB specificity between P22 and lambda N. A boxB specific for P22 N can be mutated to lambda N specificity by a series of single mutations via a bifunctional intermediate, as predicted by neutral theories of evolution.

Structural Insights Lead to a Negamycin Analogue with Improved Antimicrobial Activity against Gram-Negative Pathogens.

Negamycin is a natural product with antibacterial activity against a broad range of Gram-negative pathogens. Recent revelation of its ribosomal binding site and mode of inhibition has reinvigorated efforts to identify improved analogues with clinical potential. Translation-inhibitory potency and antimicrobial activity upon modification of different moieties of negamycin were in line with its observed ribosomal binding conformation, reaffirming stringent structural requirements for activity. However, substitutions on the N6 amine were tolerated and led to N6-(3-aminopropyl)-negamycin (31f), an analogue showing 4-fold improvement in antibacterial activity against key bacterial pathogens. This represents the most potent negamycin derivative to date and may be a stepping stone toward clinical development of this novel antibacterial class.

Structure of the Cmr2-Cmr3 subcomplex of the Cmr RNA silencing complex.

The Cmr complex is an RNA-guided effector complex that cleaves invader RNA in the prokaryotic immune response mediated by the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas system. Here, we report the crystal structure of a Cmr subcomplex containing Cmr2 (Cas10) and Cmr3 subunits at 2.8 Å resolution. The structure revealed a dual ferredoxin fold and glycine-rich loops characteristic of previously known repeat-associated mysterious proteins and two unique insertion elements in Cmr3 that mediate its interaction with Cmr2. Surprisingly, while mutation of both insertion elements significantly weakened Cmr3-Cmr2 interaction, they exhibit differential effects on Cmr-mediated RNA cleavage by the Cmr complex, suggesting stabilization of Cmr2-Cmr3 interactions by other subunits. Further mutational analysis of the two conserved (but non-Cmr2-binding) glycine-rich loops of Cmr3 identified a region that is likely involved in assembly or the RNA cleavage function of the Cmr complex.

Structure of the Cmr2 subunit of the CRISPR-Cas RNA silencing complex.

Cmr2 is the largest and an essential subunit of a CRISPR RNA-Cas protein complex (the Cmr complex) that cleaves foreign RNA to protect prokaryotes from invading genetic elements. Cmr2 is thought to be the catalytic subunit of the effector complex because of its N-terminal HD nuclease domain. Here, however, we report that the HD domain of Cmr2 is not required for cleavage by the complex in vitro. The 2.3Å crystal structure of Pyrococcus furiosus Cmr2 (lacking the HD domain) reveals two adenylyl cyclase-like and two α-helical domains. The adenylyl cyclase-like domains are arranged as in homodimeric adenylyl cyclases and bind ADP and divalent metals. However, mutagenesis studies show that the metal- and ADP-coordinating residues of Cmr2 are also not critical for cleavage by the complex. Our findings suggest that another component provides the catalytic function and that the essential role by Cmr2 does not require the identified ADP- or metal-binding or HD domains in vitro.