Structure of the Capsid Size-Determining Scaffold of “Satellite” Bacteriophage P4

P4 is a mobile genetic element (MGE) that can exist as a plasmid or integrated into its Escherichia coli host genome, but becomes packaged into phage particles by a helper bacteriophage, such as P2. P4 is the original example of what we have termed “molecular piracy”, the process by which one MGE usurps the life cycle of another for its own propagation. The P2 helper provides most of the structural gene products for assembly of the P4 virion. However, when P4 is mobilized by P2, the resulting capsids are smaller than those normally formed by P2 alone. The P4-encoded protein responsible for this size change is called Sid, which forms an external scaffolding cage around the P4 procapsids. We have determined the high-resolution structure of P4 procapsids, allowing us to build an atomic model for Sid as well as the gpN capsid protein. Sixty copies of Sid form an intertwined dodecahedral cage around the T = 4 procapsid, making contact with only one out of the four symmetrically non-equivalent copies of gpN. Our structure provides a basis for understanding the sir mutants in gpN that prevent small capsid formation, as well as the nms “super-sid” mutations that counteract the effect of the sir mutations, and suggests a model for capsid size redirection by Sid.

A Bacteriophage Capsid Protein Is an Inhibitor of a Conserved Transcription Terminator of Various Bacterial Pathogens

ABSTRACTRho is a hexameric molecular motor that functions as a conserved transcription terminator in the majority of bacterial species and is a potential drug target. Psu is a bacteriophage P4 capsid protein that inhibitsEscherichia coliRho by obstructing its ATPase and translocase activities. In this study, we explored the anti-Rho activity of Psu for Rho proteins from different pathogens. Sequence alignment and homology modeling of Rho proteins from pathogenic bacteria revealed the conserved nature of the Psu-interacting regions in all these proteins. We chose Rho proteins from various pathogens, includingMycobacterium smegmatis,Mycobacterium bovis,Mycobacterium tuberculosis,Xanthomonas campestris,Xanthomonas oryzae,Corynebacterium glutamicum,Vibrio cholerae,Salmonella enterica, andPseudomonas syringae. The purified recombinant Rho proteins of these organisms showed variable rates of ATP hydrolysis on poly(rC) as the substrate and were capable of releasing RNA from theE. colitranscription elongation complexes. Psu was capable of inhibiting these two functions of all these Rho proteins.In vivopulldown assays revealed direct binding of Psu with many of these Rho proteins.In vivoexpression ofpsuinduced killing ofM. smegmatis,M. bovis,X. campestris, andE. coliexpressingS. entericaRho indicating Psu-induced inhibition of Rho proteins of these strains under physiological conditions. We propose that the “universal” inhibitory function of the Psu protein against the Rho proteins from both Gram-negative and Gram-positive bacteria could be useful for designing peptides with antimicrobial functions and that these peptides could contribute to synergistic antibiotic treatment of the pathogens by compromising the Rho functions.IMPORTANCEBacteriophage-derived protein factors modulating different bacterial processes could be converted into unique antimicrobial agents. Bacteriophage P4 capsid protein Psu is an inhibitor of theE. colitranscription terminator Rho. Here we show that apart from antagonizingE. coliRho, Psu is able to inhibit Rho proteins from various phylogenetically unrelated Gram-negative and Gram-positive pathogens. Upon binding to these Rho proteins, Psu inhibited them by affecting their ATPase and RNA release functions. The expression of Psuin vivokills various pathogens, such asMycobacteriumandXanthomonasspecies. Hence, Psu could be useful for identifying peptide sequences with anti-Rho activities and might constitute part of synergistic antibiotic treatment against pathogens.

The Knot Spectrum of Confined Random Equilateral Polygons

It is well known that genomic materials (long DNA chains) of living organisms are often packed compactly under extreme confining conditions using macromolecular self-assembly processes but the general DNA packing mechanism remains an unsolved problem. It has been proposed that the topology of the packed DNA may be used to study the DNA packing mechanism. For example, in the case of (mutant) bacteriophage P4, DNA molecules packed inside the bacteriophage head are considered to be circular since the two sticky ends of the DNA are close to each other. The DNAs extracted from the capsid without separating the two ends can thus preserve the topology of the (circular) DNAs. It turns out that the circular DNAs extracted from bacteriophage P4 are non-trivially knotted with very high probability and with a bias toward chiral knots. In order to study this problem using a systematic approach based on mathematical modeling, one needs to introduce a DNA packing model under extreme volume confinement condition and test whether such a model can produce the kind of knot spectrum observed in the experiments. In this paper we introduce and study a model of equilateral random polygons con_ned in a sphere. This model is not meant to generate polygons that model DNA packed in a virus head directly. Instead, the average topological characteristics of this model may serve as benchmark data for totally randomly packed circular DNAs. The difference between the biologically observed topological characteristics and our benchmark data might reveal the bias of DNA packed in the viral capsids and possibly lead to a better understanding of the DNA packing mechanism, at least for the bacteriophage DNA. The purpose of this paper is to provide information about the knot spectrum of equilateral random polygons under such a spherical confinement with length and confinement ratios in a range comparable to circular DNAs packed inside bacteriophage heads.