Structural modeling and functional analysis of the essential ribosomal processing protease Prp from Staphylococcus aureus

In Firmicutes and related bacteria, ribosomal large subunit protein L27 is encoded with a conserved N-terminal extension that is removed to expose residues critical for ribosome function. Bacteria encoding L27 with this N-terminal extension also encode a sequence-specific cysteine protease, Prp, which carries out this cleavage. In this work, we demonstrate that L27 variants with an un-cleavable N-terminal extension, or lacking the extension (pre-cleaved), are unable to complement an L27 deletion in Staphylococcus aureus. This indicates that N-terminal processing of L27 is not only essential but possibly has a regulatory role. Prp represents a new clade of previously uncharacterized cysteine proteases, and the dependence of S. aureus on L27 cleavage by Prp validates the enzyme as a target for potential antibiotic development. To better understand the mechanism of Prp activity, we analyzed Prp enzyme kinetics and substrate preference using a fluorogenic peptide cleavage assay. Molecular modeling and site-directed mutagenesis implicate several residues around the active site in catalysis and substrate binding, and support a structural model in which rearrangement of a flexible loop upon binding of the correct peptide substrate is required for the active site to assume the proper conformation. These findings lay the foundation for the development of antimicrobials that target this novel, essential pathway

Mapping the packaging cleavage determinants in the phage-dependent Staphylococcus aureus pathogenicity island SaPI1

Members of the SaPI family of Staphylococcus aureus pathogenicity islands are highly mobile, superantigen-encoding genetic elements that depend upon specific helper bacteriophages for their horizontal transfer. Following helper phage infection, these elements excise, replicate, and package their genomes in virions provided by the helper phage. One of the novel features of this process is the use of a SaPI-encoded small subunit of terminase, which replaces the phage small terminase subunit and redirects terminase packaging specificity to SaPI DNA. The SaPI genome, like that of the helper phage, is packaged from concatemers by a headful packaging mechanism, resulting in encapsidated DNA molecules that exhibit terminal redundancy and partial circular permutation. In this study we have localized the site of initial cleavage in SaPI1, a prototype member of this family, and have defined a small region of the SaPI1 genome sufficient for specific packaging. The cleavage site was initially localized to an intergenic region upstream of SaPI1 operon 1 by identification of submolar fragments in restricted SaPI1 virion DNA. Ligation of linkers to the ends of SaPI virion DNA, followed by amplification and sequencing of the linker/SaPI1 junction, further defined the sites of initial cleavage. A fragment encompassing this region was cloned into a plasmid vector co-expressing SaPI1 small terminase and shown to confer high frequency plasmid transduction by helper phage 80α. The critical determinants for SaPI1-specific packaging were further defined by deletion analysis of the cloned fragment. These results demonstrate that the sequence required for SaPI1-specific packaging maps to a small region upstream of the promoter for SaPI1 operon 1. This is strikingly different from the pac site used by the helper phage terminase, which, like other phage pac sites, maps to within the small terminase gene itself

Sequence determinants for DNA packaging specificity in the S. aureus pathogenicity island SaPI1

The SaPIs and their relatives are a family of genomic islands that exploit helper phages for high frequency horizontal transfer. One of the mechanisms used by SaPIs to accomplish this molecular piracy is the redirection of the helper phage DNA packaging machinery. SaPIs encode a small terminase subunit that can be substituted for that of the phage. In this study we have determined the initial packaging cleavage sites for helper phage 80α, which uses the phage-encoded small terminase subunit, and for SaPI1, which uses the SaPI-encoded small terminase subunit. We have identified a 19 nt SaPI1 sequence that is necessary and sufficient to allow high frequency 80α transduction of a plasmid by a terminase carrying the SaPI1-encoded small subunit. We also show that the hybrid enzyme with the SaPI1 small terminase subunit is capable of generalized transduction.This work was performed in part under FDA's Medical Countermeasures Initiative, Contract #HHSF2232010000521 and NIH 1R56 AI081837 (to GEC). JCB was supported by the Portuguese Institute Fundacao para a Ciencia e Tecnologia (FCT), PhD Fellowship SFRH/BD/66250/2009. E.K.R. was supported by the National Academies National Research Council; his contribution to this study was carried out at NICHHD, NIH, Bethesda, MD, in the laboratory of the late Robert A. Weisberg. Plasmid pCN51 (in strain NRS613) and strain RN4282 (NRS145) were obtained through the Network of Antimicrobial Resistance in Staphylococcus aureus (NARSA) program supported under NIAID/NIFI contract #HHSN272200700055C

A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria

The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the ter S gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria.Funding for open access charge: Ministerio de Ciencia e Innovación (MICINN) [Consolider-Ingenio CSD2009-00006, BIO2011-30503-C02-01 and Eranet-pathogenomics PIM2010EPA-00606 to J.R.P]; Cardenal Herrera-CEU University [Copernicus-Santander program to J.R.P.]; Insituto Nacional de Investigaciones Agrarias (INIA) [DR08-0093 to M.A.T-M.]; National Institute of Health [R56AI081837 to G.E.C, R01AI022159-23A2 to R.P.N.]

Fungal BLAST and Model Organism BLASTP Best Hits: new comparison resources at the Saccharomyces Genome Database (SGD)

The Saccharomyces Genome Database (SGD; http://www.yeastgenome.org/) is a scientific database of gene, protein and genomic information for the yeast Saccharomyces cerevisiae. SGD has recently developed two new resources that facilitate nucleotide and protein sequence comparisons between S.cerevisiae and other organisms. The Fungal BLAST tool provides directed searches against all fungal nucleotide and protein sequences available from GenBank, divided into categories according to organism, status of completeness and annotation, and source. The Model Organism BLASTP Best Hits resource displays, for each S.cerevisiae protein, the single most similar protein from several model organisms and presents links to the database pages of those proteins, facilitating access to curated information about potential orthologs of yeast proteins

RinA controls phage-mediated packaging and transfer of virulence genes in Gram-positive bacteria

Phage-mediated transfer of microbial genetic elements plays a crucial role in bacterial life style and evolution. In this study, we identify the RinA family of phage-encoded proteins as activators required for transcription of the late operon in a large group of temperate staphylococcal phages. RinA binds to a tightly regulated promoter region, situated upstream of the terS gene, that controls expression of the morphogenetic and lysis modules of the phage, activating their transcription. As expected, rinA deletion eliminated formation of functional phage particles and significantly decreased the transfer of phage and pathogenicity island encoded virulence factors. A genetic analysis of the late promoter region showed that a fragment of 272 bp contains both the promoter and the region necessary for activation by RinA. In addition, we demonstrated that RinA is the only phage-encoded protein required for the activation of this promoter region. This region was shown to be divergent among different phages. Consequently, phages with divergent promoter regions carried allelic variants of the RinA protein, which specifically recognize its own promoter sequence. Finally, most Gram-postive bacteria carry bacteriophages encoding RinA homologue proteins. Characterization of several of these proteins demonstrated that control by RinA of the phage-mediated packaging and transfer of virulence factor is a conserved mechanism regulating horizontal gene transfer

Expanded protein information at SGD: new pages and proteome browser

The recent explosion in protein data generated from both directed small-scale studies and large-scale proteomics efforts has greatly expanded the quantity of available protein information and has prompted the Saccharomyces Genome Database (SGD; ) to enhance the depth and accessibility of protein annotations. In particular, we have expanded ongoing efforts to improve the integration of experimental information and sequence-based predictions and have redesigned the protein information web pages. A key feature of this redesign is the development of a GBrowse-derived interactive Proteome Browser customized to improve the visualization of sequence-based protein information. This Proteome Browser has enabled SGD to unify the display of hidden Markov model (HMM) domains, protein family HMMs, motifs, transmembrane regions, signal peptides, hydropathy plots and profile hits using several popular prediction algorithms. In addition, a physico-chemical properties page has been introduced to provide easy access to basic protein information. Improvements to the layout of the Protein Information page and integration of the Proteome Browser will facilitate the ongoing expansion of sequence-specific experimental information captured in SGD, including post-translational modifications and other user-defined annotations. Finally, SGD continues to improve upon the availability of genetic and physical interaction data in an ongoing collaboration with BioGRID by providing direct access to more than 82 000 manually-curated interactions

Genome Snapshot: a new resource at the Saccharomyces Genome Database (SGD) presenting an overview of the Saccharomyces cerevisiae genome

Sequencing and annotation of the entire Saccharomyces cerevisiae genome has made it possible to gain a genome-wide perspective on yeast genes and gene products. To make this information available on an ongoing basis, the Saccharomyces Genome Database (SGD) () has created the Genome Snapshot (). The Genome Snapshot summarizes the current state of knowledge about the genes and chromosomal features of S.cerevisiae. The information is organized into two categories: (i) number of each type of chromosomal feature annotated in the genome and (ii) number and distribution of genes annotated to Gene Ontology terms. Detailed lists are accessible through SGD's Advanced Search tool (), and all the data presented on this page are available from the SGD ftp site ()

Do state and trait measures measure states and traits? The case of community-dwelling caregivers of older adults

Spielberger’s state and trait anxiety and anger scales are widely used and documented, but there is little or no direct evidence that they actually measure their respective state and trait aspects as was intended. We conducted latent state-trait analyses on data collected from 310 community-dwelling caregivers of older adult care recipients and found that (a) both state and trait scales reflected a mixture of state and trait aspects of their latent constructs, (b) state scales reflected more state-like variance than did corresponding trait scales, but (c) both state and trait scales were dominated by stable trait-like variance. Follow-up bivariate latent state-trait analyses indicated that correlations between trait components of anger and anxiety correlated more strongly with trait components of caregiver–care recipient mutually communal behavior and care recipient problem behavior than did state–state component correlations. Implications for the measurement of state and trait components of psychological constructs are discussed