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A Novel Putative Enterococcal Pathogenicity Island Linked to the esp Virulence Gene of Enterococcus faecium and Associated with Epidemicity. Helen Leavis, 2004.Enterococcus faecalis harbors a virulence-associated surface protein encoded by the esp gene . This gene has been shown to be part of a 150-kb putative pathogenicity island . A gene similar to esp has recently been found in Enterococcus faecium isolates recovered from hospitalized patients . In the present study we analyzed the polymorphism in the esp gene of E . faecium, and we investigated the association of esp with neighboring chromosomal genes . The esp gene showed considerable sequence heterogeneity in the regions encoding the nonrepeat N- and C-terminal domains of the Esp protein as well as differences in the number of repeats . DNA sequencing of chromosomal regions flanking the esp gene of E . faecium revealed seven open reading frames, representing putative genes implicated in virulence, regulation of transcription, and antibiotic resistance . These flanking regions were invariably associated with the presence or absence of the esp gene in E . faecium, indicating that esp in E . faecium is part of a distinct genetic element . Because of the presence of virulence genes in this gene cluster, the lower G+C content relative to that of the genome, and the presence of esp in E . faecium isolates associated with nosocomial outbreaks and clinically documented infections, we conclude that this genetic element constitutes a putative pathogenicity island, the first one described in E . faecium . Except for the presence of esp and araC, this pathogenicity island is completely different from the esp-containing pathogenicity island previously disclosed in E . faecalis. Role of a Mutation at Position 167 of CTX-M-19 in Ceftazidime Hydrolysis. Soichiro Kimura, 2004.CTX-M-19 is a recently identified ceftazidime-hydrolyzing extended-spectrum ß-lactamase, which differs from the majority of CTX-M-type ß-lactamases that preferentially hydrolyze cefotaxime but not ceftazidime . To elucidate the mechanism of ceftazidime hydrolysis by CTX-M-19, the ß-lactam MICs of a CTX-M-19 producer, and the kinetic parameters of the enzyme were confirmed . We reconfirmed here that CTX-M-19 is also stable at a high enzyme concentration in the presence of bovine serum albumin (20 µg/ml) . Under this condition, we obtained more accurate kinetic parameters and determined that cefotaxime (kcat/Km, 1.47 x 106 s–1 M–1), cefoxitin (kcat/Km, 62.2 s–1 M–1), and aztreonam (kcat/Km, 1.34 x 103 s–1 M–1) are good substrates and that imipenem (k+2/K, 1.20 x 102 s–1 M–1) is a poor substrate . However, CTX-M-18 and CTX-M-19 exhibited too high a Km value (2.7 to 5.6 mM) against ceftazidime to obtain their catalytic activity (kcat) . Comparison of the MICs with the catalytic efficiency (kcat/Km) of these enzymes showed that some ß-lactams, including cefotaxime, ceftazidime, and aztreonam showed a similar correlation . Using the previously reported crystal structure of the Toho-1 ß-lactamase, which belongs to the CTX-M-type ß-lactamase group, we have suggested characteristic interactions between the enzymes and the ß-lactams ceftazidime, cefotaxime, and aztreonam by molecular modeling . Aminothiazole-bearing ß-lactams require a displacement of the aminothiazole moiety due to a severe steric interaction with the hydroxyl group of Ser167 in CTX-M-19, and the displacement affects the interaction between Ser130 and the acidic group such as carboxylate and sulfonate of ß-lactams . Nanoscale Investigation of Pathogenic Microbial Adhesion to a Biomaterial. Ray J. Emerson IV, 2004.Microbial infections of medical implants occur in more than 2 million surgical cases each year in the United States alone . These increase patient morbidity and mortality, as well as patient cost and recovery time . Many treatments are available, but none are guaranteed to remove the infection . In many cases, the device infections are caused by the adhesion of microbes to the implant, ensuing growth, pathogenesis, and dissemination . The purpose of this work is to examine the initial events in microbial adhesion by simulating the approach and contact between a planktonic cell, immobilized on an atomic force microscope (AFM) cantilever, and a biomaterial or biofilm substrate . The two model microbes used in this study, Candida parapsilosis (ATCC 90018) and Pseudomonas aeruginosa (ATCC 10145), were chosen for both their clinical relevance and their ease of acquisition and handling in the laboratory setting . Attractive interactions exist between C . parapsilosis and both unmodified silicone rubber and P . aeruginosa biofilms . Using C . parapsilosis cells immobilized on AFM cantilevers with a silicone substrate, we have measured attractive forces of 4.3 ± 0.25 nN in the approach portion of the force cycle . On P . aeruginosa biofilms, the magnitude of the attractive force decreases to 2.0 ± 0.40 nN and is preceded by a 2.0-nN repulsion at approximately 75 nm from the cell surface . These data suggest that C . parapsilosis may adhere to both silicone rubber and P . aeruginosa biofilms, possibly contributing to patient morbidity and mortality . Characterization of cell-biomaterial and cell-cell interactions allows for a quantitative link between the physicomechanical and physicochemical properties of implant materials and the nanoscale interactions leading to microbial colonization and infection . The Phage Proteomic Tree: a Genome-Based Taxonomy for Phage. Forest Rohwer, 2002.There are  1031 phage in the biosphere, making them the most abundant biological entities on the planet . Despite their great numbers and ubiquitous presence, very little is known about phage biodiversity, biogeography, or phylogeny . Information is limited, in part, because the current ICTV taxonomical system is based on culturing phage and measuring physical parameters of the free virion . No sequence-based taxonomic systems have previously been established for phage . We present here the "Phage Proteomic Tree," which is based on the overall similarity of 105 completely sequenced phage genomes . The Phage Proteomic Tree places phage relative to both their near neighbors and all other phage included in the analysis . This method groups phage into taxa that predicts several aspects of phage biology and highlights genetic markers that can be used for monitoring phage biodiversity . We propose that the Phage Proteomic Tree be used as the basis of a genome-based taxonomical system for phage .
Molecular Analysis of Phr Peptide Processing in Bacillus subtilis. Sophie Stephenson, 2003.In Bacillus subtilis, an export-import pathway regulates production of the Phr pentapeptide inhibitors of Rap proteins . Processing of the Phr precursor proteins into the active pentapeptide form is a key event in the initiation of sporulation and competence development . The PhrA (ARNQT) and PhrE (SRNVT) peptides inhibit the RapA and RapE phosphatases, respectively, whose activity is directed toward the Spo0F  P intermediate response regulator of the sporulation phosphorelay . The PhrC (ERGMT) peptide inhibits the RapC protein acting on the ComA response regulator for competence with regard to DNA transformation . The structural organization of PhrA, PhrE, and PhrC suggested a role for type I signal peptidases in the processing of the Phr preinhibitor, encoded by the phr genes, into the proinhibitor form . The proinhibitor was then postulated to be cleaved to the active pentapeptide inhibitor by an additional enzyme . In this report, we provide evidence that Phr preinhibitor proteins are subject to only one processing event at the peptide bond on the amino-terminal end of the pentapeptide . This processing event is most likely independent of type I signal peptidase activity . In vivo and in vitro analyses indicate that none of the five signal peptidases of B . subtilis (SipS, SipT, SipU, SipV, and SipW) are indispensable for Phr processing . However, we show that SipV and SipT have a previously undescribed role in sporulation, competence, and cell growth .
Molecular Genetic Analysis of a Group A Streptococcus Operon Encoding Serum Opacity Factor and a Novel Fibronectin-Binding Protein, SfbX. Arthur Jeng, 2003.The group A Streptococcus (GAS) sof gene encodes the serum opacity factor protein, which is capable of opacifying mammalian sera and binding at least two host proteins, fibronectin and fibrinogen . The sof gene exists in approximately 50% of clinical isolates, and there is a classical association of so-called nephritogenic strains with the opacity factor-positive phenotype . In both a type emm49 strain and a type emm12 strain, the sequences upstream of the 5' end of sof and downstream of the putative terminator were determined to be nearly identical to a region in the M type 1 genome approximately 10 kb upstream of the emm1 gene . This close genetic linkage is likely reflected in the strict correlation of opacity factor phenotype with specific emm genotypes . A new fibronectin-binding protein gene, sfbX, was discovered immediately downstream of sof in emm12 and emm49 strains and in several other sof-positive strains . The sof and sfbX genes were found to be expressed on the same transcription unit, which was correlated with the putative promoter and rho-independant terminator sequences that flank these two genes . The sfbX genes from different emm types are predicted to encode  650-residue surface-bound proteins sharing 89 to 92% sequence identity . SfbX residues approximately 1 to 480 are not highly similar to those of other known proteins, with the closest match being the Staphylococcus aureus coagulase protein . The remaining portions of these proteins (residues 481 to 650) contain four putative fibronectin-binding repeats highly similar to those of other streptococcal fibronectin-binding proteins and a potential LP(X)SG cell wall anchor motif . Targeted in-frame allelic-exchange mutagenesis, complementation, and heterologous-expression studies found that serum opacification is encoded by sof alone and that sfbX encodes a fibronectin-binding function . A recombinant SfbX protein was found to bind immobilized fibronectin and to partially inhibit GAS adherence to fibronectin . The sfbX gene was found to be present only in sof-positive strains, and together these genes could influence the spectrum of tissues colonized by sof-positive GAS .
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