bluuebunny/crossref_metadata_2025 huggingface dataset:
Substrate and Inhibitor Spectra of Ethylbenzene Dehydrogenase: Perspectives on Application Potential and Catalytic Mechanism
Authors: Knack Daniel , Hagel Corina , Szaleniec Maciej , Dudzik Agnieszka , Salwinski Aleksander , Heider Johann
Published: July 2012

Ethylbenzene dehydrogenase (EbDH) catalyzes the initial step in anaerobic degradation of ethylbenzene in denitrifying bacteria, namely, the oxygen-independent hydroxylation of ethylbenzene to (
S
)-1-phenylethanol. In our study we investigate the kinetic properties of 46 substrate analogs acting as substrates or inhibitors of the enzyme. The apparent kinetic parameters of these compounds give important insights into the function of the enzyme and are consistent with the predicted catalytic mechanism based on a quantum chemical calculation model. In particular, the existence of the proposed substrate-derived radical and carbocation intermediates is substantiated by the formation of alternative dehydrogenated and hydroxylated products from some substrates, which can be regarded as mechanistic models. In addition, these results also show the surprisingly high diversity of EbDH in hydroxylating different kinds of alkylaromatic and heterocyclic compounds to the respective alcohols. This may lead to attractive industrial applications of ethylbenzene dehydrogenase for a new process of producing alcohols via hydroxylation of the corresponding aromatic hydrocarbons rather than the customary procedure of reducing the corresponding ketones.
Genetic Diversity and Mating Type Distribution of Tuber melanosporum and Their Significance to Truffle Cultivation in Artificially Planted Truffiéres in Australia
Authors: Linde C. C. , Selmes H.
Published: July 2012

Tuber melanosporum
is a truffle native to Europe and is cultivated in countries such as Australia for the gastronomic market, where production yields are often lower than expected. We assessed the genetic diversity of
T. melanosporum
with six microsatellite loci to assess the effect of genetic drift on truffle yield in Australia. Genetic diversity as assessed on 210 ascocarps revealed a higher allelic diversity compared to previous studies from Europe, suggesting a possible genetic expansion and/or multiple and diverse source populations for inoculum. The results also suggest that the single sequence repeat diversity of locus ME2 is adaptive and that, for example, the probability of replication errors is increased for this locus. Loss of genetic diversity in Australian populations is therefore not a likely factor in limiting ascocarp production. A survey of nursery seedlings and trees inoculated with
T. melanosporum
revealed that <70% of seedlings and host trees were colonized with
T. melanosporum
and that some trees had been contaminated by
Tuber brumale
, presumably during the inoculation process. Mating type (MAT1-1-1 and MAT1-2-1) analyses on seedling and four- to ten-year-old host trees found that 100% of seedlings but only approximately half of host trees had both mating types present. Furthermore, MAT1-1-1 was detected significantly more commonly than MAT1-2-1 in established trees, suggesting a competitive advantage for MAT1-1-1 strains. This study clearly shows that there are more factors involved in ascocarp production than just the presence of both mating types on host trees.
Exploring the Natural Fungal Biodiversity of Tropical and Temperate Forests toward Improvement of Biomass Conversion
Authors: Berrin Jean-Guy , Navarro David , Couturier Marie , Olivé Caroline , Grisel Sacha , Haon Mireille , Taussac Sabine , Lechat Christian , Courtecuisse Régis , Favel Anne , Coutinho Pedro M. , Lesage-Meessen Laurence
Published: July 2012

In this study, natural fungal diversity in wood-decaying species was explored for biomass deconstruction. In 2007 and 2008, fungal isolates were collected in temperate forests mainly from metropolitan France and in tropical forests mainly from French Guiana. We recovered and identified 74 monomorph cultures using morphological and molecular identification tools. Following production of fungal secretomes under inductive conditions, we evaluated the capacity of these fungal strains to potentiate a commercial
Trichoderma reesei
cellulase cocktail for the release of soluble sugars from biomass. The secretome of 19 isolates led to an improvement in biomass conversion of at least 23%. Of the isolates, the
Trametes gibbosa
BRFM 952 (Banque de Ressources Fongiques de Marseille) secretome performed best, with 60% improved conversion, a feature that was not universal to the
Trametes
and related genera. Enzymatic characterization of the
T. gibbosa
BRFM 952 secretome revealed an unexpected high activity on crystalline cellulose, higher than that of the
T. reesei
cellulase cocktail. This report highlights the interest in a systematic high-throughput assessment of collected fungal biodiversity to improve the enzymatic conversion of lignocellulosic biomass. It enabled the unbiased identification of new fungal strains issued from biodiversity with high biotechnological potential.
Lack of Cry1Fa Binding to the Midgut Brush Border Membrane in a Resistant Colony of Plutella xylostella Moths with a Mutation in the
ABCC2
Locus
Authors: Hernández-Martínez Patricia , Hernández-Rodríguez Carmen Sara , Krishnan Vidisha , Crickmore Neil , Escriche Baltasar , Ferré Juan
Published: July 2012

Previous studies reported “mode 1”
Bacillus thuringiensis
resistance in a colony of diamondback moths (NO-QA), and recently, this resistance has been mapped to an ABC transporter (
ABCC2
) locus. We report the lack of binding of Cry1Fa to insects derived from this colony and compare our data with those from other insects with
ABCC2
-associated resistance.
Structure-Guided Expansion of the Substrate Range of Methylmalonyl Coenzyme A Synthetase (MatB) of Rhodopseudomonas palustris
Authors: Crosby Heidi A. , Rank Katherine C. , Rayment Ivan , Escalante-Semerena Jorge C.
Published: July 2012

Malonyl coenzyme A (malonyl-CoA) and methylmalonyl-CoA are two of the most commonly used extender units for polyketide biosynthesis and are utilized to synthesize a vast array of pharmaceutically relevant products with antibacterial, antiparasitic, anticholesterol, anticancer, antifungal, and immunosuppressive properties. Heterologous hosts used for polyketide production such asEscherichia colioften do not produce significant amounts of methylmalonyl-CoA, however, requiring the introduction of other pathways for the generation of this important building block. Recently, the bacterial malonyl-CoA synthetase class of enzymes has been utilized to generate malonyl-CoA and methylmalonyl-CoA directly from malonate and methylmalonate. We demonstrate that in the purple photosynthetic bacteriumRhodopseudomonas palustris, MatB (RpMatB) acts as a methylmalonyl-CoA synthetase and is required for growth on methylmalonate. We report theapo(1.7-Å resolution) and ATP-bound (2.0-Å resolution) structure and kinetic analysis ofRpMatB, which shows similar activities for both malonate and methylmalonate, making it an ideal enzyme for heterologous polyketide biosynthesis. Additionally, rational, structure-based mutagenesis of the active site ofRpMatB led to substantially higher activity with ethylmalonate and butylmalonate, demonstrating that this enzyme is a prime target for expanded substrate specificity.
Comparative Analysis of Mycobacterial Truncated Hemoglobin Promoters and thegroEL2Promoter in Free-Living and Intracellular Mycobacteria
Authors: Joseph Sunil V. , Madhavilatha G. K. , Kumar R. Ajay , Mundayoor Sathish
Published: July 2012

The success ofMycobacterium tuberculosisdepends on its ability to withstand and survive the hazardous environment inside the macrophages that are created by reactive oxygen intermediates, reactive nitrogen intermediates, severe hypoxia, low pH, and high CO2levels. Therefore, an effective detoxification system is required for the pathogen to persistin vivo. The genome ofM. tuberculosiscontains a new family of hemoproteins named truncated hemoglobin O (trHbO) and truncated hemoglobin N (trHbN), encoded by theglbOandglbNgenes, respectively, important in the survival ofM. tuberculosisin macrophages. Mycobacterial heat shock proteins are known to undergo rapid upregulation under stress conditions. The expression profiles of the promoters of these genes were studied by constructing transcriptional fusions with green fluorescent protein and monitoring the promoter activity in both free-living and intracellular milieus at different time points. WhereasglbNshowed an early response to the oxidative and nitrosative stresses tested,glbOgave a lasting response to lower concentrations of both stresses. At all time points and under all stress conditions tested,groEL2showed higher expression than both trHb promoters and expression of both promoters showed an increase while inside the macrophages. Real-time PCR analysis of trHb andgroEL2mRNAs showed an initial upregulation at 24 h postinfection. The presence of theglbOprotein imparted an increased survival toM. smegmatisin THP-1 differentiated macrophages compared to that imparted by theglbNandhsp65proteins. The comparative upregulation shown by both trHb promoters while grown inside macrophages indicates the importance of these promoters for the survival ofM. tuberculosisin the hostile environment of the host.
Pediatric Invasive Pneumococcal Disease in the United States in the Era of Pneumococcal Conjugate Vaccines
Authors: Tan Tina Q.
Published: July 2012

Invasive infections caused by
Streptococcus pneumoniae
continue to be a major cause of morbidity and mortality worldwide, especially in children under 5 years of age. In the United States, 90% of invasive pneumococcal infections in children are caused by 13 serotypes of
S. pneumoniae
. The licensure (in 2000) and subsequent widespread use of a heptavalent pneumococcal conjugate vaccine (PCV7) have had a significant impact on decreasing the incidence of serious invasive pneumococcal disease (IPD) in all age groups, especially in children under 2 years of age. However, the emergence of replacement non-PCV7 serotypes, especially serotype 19A, has resulted in an increase in the incidence of serious and invasive infections. In 2010, a 13-valent PCV was licensed in the United States. However, the impact that this vaccine will have on IPD remains to be seen. The objectives of this review are to discuss the epidemiology of serious and invasive pneumococcal infections in the United States in the PCV era and to review some of the pneumococcal vaccines that are in development.
Antimicrobial Susceptibility Testing, Drug Resistance Mechanisms, and Therapy of Infections with Nontuberculous Mycobacteria
Authors: Brown-Elliott Barbara A. , Nash Kevin A. , Wallace Richard J.
Published: July 2012

Within the past 10 years, treatment and diagnostic guidelines for nontuberculous mycobacteria have been recommended by the American Thoracic Society (ATS) and the Infectious Diseases Society of America (IDSA). Moreover, the Clinical and Laboratory Standards Institute (CLSI) has published and recently (in 2011) updated recommendations including suggested antimicrobial and susceptibility breakpoints. The CLSI has also recommended the broth microdilution method as the gold standard for laboratories performing antimicrobial susceptibility testing of nontuberculous mycobacteria. This article reviews the laboratory, diagnostic, and treatment guidelines together with established and probable drug resistance mechanisms of the nontuberculous mycobacteria.
Enteric Protozoa in the Developed World: a Public Health Perspective
Authors: Fletcher Stephanie M. , Stark Damien , Harkness John , Ellis John
Published: July 2012

Several enteric protozoa cause severe morbidity and mortality in both humans and animals worldwide. In developed settings, enteric protozoa are often ignored as a cause of diarrheal illness due to better hygiene conditions, and as such, very little effort is used toward laboratory diagnosis. Although these protozoa contribute to the high burden of infectious diseases, estimates of their true prevalence are sometimes affected by the lack of sensitive diagnostic techniques to detect them in clinical and environmental specimens. Despite recent advances in the epidemiology, molecular biology, and treatment of protozoan illnesses, gaps in knowledge still exist, requiring further research. There is evidence that climate-related changes will contribute to their burden due to displacement of ecosystems and human and animal populations, increases in atmospheric temperature, flooding and other environmental conditions suitable for transmission, and the need for the reuse of alternative water sources to meet growing population needs. This review discusses the common enteric protozoa from a public health perspective, highlighting their epidemiology, modes of transmission, prevention, and control. It also discusses the potential impact of climate changes on their epidemiology and the issues surrounding waterborne transmission and suggests a multidisciplinary approach to their prevention and control.
Sensitivity and Specificity of an Operon Immunochromatographic Test in Serum and Whole-Blood Samples for the Diagnosis of Trypanosoma cruzi Infection in Spain, an Area of Nonendemicity
Authors: Flores-Chavez María , Cruz Israel , Nieto Javier , Gárate Teresa , Navarro Miriam , Pérez-Ayala Ana , López-Vélez Rogelio , Cañavate Carmen
Published: July 2012

Trypanosoma cruzi
infection is an imported parasitic disease in Spain, and the majority of infected individuals are in the chronic phase of the disease. This study evaluated the sensitivity and specificity of the Operon immunochromatographic test (ICT-Operon; Simple Stick Chagas and Simple Chagas WB [whole blood]; Operon S.A., Spain) for different biological samples. Well-characterized serum samples were obtained from chagasic patients (
n
= 63), nonchagasic individuals (
n
= 95), visceral leishmaniasis patients (
n
= 38), and malaria patients (
n
= 55). Noncharacterized specimens were obtained from Latin American immigrants and individuals at risk with a clinical and/or epidemiological background: these specimens were recovered serum or plasma samples (
n
= 450), whole peripheral blood (
n
= 94), and capillary blood (
n
= 282). The concordance of the results by enzyme-linked immunosorbent assay and indirect immunofluorescence test was considered to be the “gold standard” for diagnosis. Serum and plasma samples were analyzed by Stick Chagas, and whole blood was analyzed by Simple Chagas WB. The sensitivity and specificity of the ICT-Operon in well-characterized samples were 100% and 97.9%, respectively. No cross-reactivity was found with samples obtained from visceral leishmaniasis patients. In contrast, a false-positive result was obtained in 27.3% of samples from malaria patients. The sensitivities of the rapid test in noncharacterized serum or plasma, peripheral blood, and capillary blood samples were 100%, 92.1%, and 86.4%, respectively, while the specificities were 91.6%, 93.6%, and 95% in each case. ICT-Operon showed variable sensitivity, depending on the kind of sample, performing better when serum or plasma samples were used. It could therefore be used for serological screening combined with any other conventional test.
Goats Primed with Mycobacterium bovis BCG and Boosted with a Recombinant Adenovirus Expressing Ag85A Show Enhanced Protection against Tuberculosis
Authors: Pérez de Val Bernat , Villarreal-Ramos Bernardo , Nofrarías Miquel , López-Soria Sergio , Romera Nadine , Singh Mahavir , Abad F. Xavier , Xing Zhou , Vordermeier H. Martin , Domingo Mariano
Published: July 2012

This is the first efficacy study using the experimental goat model, a natural host of tuberculosis (TB), to evaluate the efficacy of heterologousMycobacterium bovisbacillus Calmette-Guérin (BCG) prime followed by boosting with a replication-deficient adenovirus expressing the antigen Ag85A (AdAg85A). Three experimental groups of 11 goat kids each were used: BCG vaccinated, BCG vaccinated and AdAg85A boosted, and nonvaccinated. Twenty-two goat kids were vaccinated with ∼5 × 105CFU of BCG (week 0), and 11 of them were boosted at week 8 with 109PFU of AdAg85A. At week 14, all goats were challenged by the endobronchial route with ∼1.5 × 103CFU ofMycobacterium caprae. The animals were euthanized at week 28. Cellular and humoral immunity induced by vaccination andM. capraeinfection was measured throughout the study. After challenge BCG-AdAg85A-vaccinated animals exhibited reduced pathology compared to BCG-vaccinated animals in lungs and in pulmonary lymph nodes. There were significant reductions in bacterial load in both groups of vaccinated goats, but the reduction was more pronounced in prime-boosted animals. Antigen-specific gamma interferon (IFN-γ) and humoral responses were identified as prognostic biomarkers of vaccination outcome depending on their correlation with pathological and bacteriological results. As far as we know, this is the first report using multidetector computed tomography (MDCT) to measure vaccine efficacy against pulmonary TB in an animal model. The use in vaccine trials of animals that are natural hosts of TB may improve research into human TB vaccines.
Lipopolysaccharide Heterogeneity in the Atypical Group of Novel Emerging Brucella Species
Authors: Zygmunt Michel S. , Jacques Isabelle , Bernardet Nelly , Cloeckaert Axel
Published: July 2012

Recently, novel
Brucella
strains with phenotypic characteristics that were atypical for strains belonging to the genus
Brucella
have been reported. Phenotypically many of these strains were initially misidentified as
Ochrobactrum
spp. Two novel species have been described so far for these strains, i.e.,
B. microti
and
B. inopinata
, and other strains genetically related to
B. inopinata
may constitute other novel species as well. In this study, we analyzed the lipopolysaccharides (LPS) (smooth LPS [S-LPS] and rough LPS [R-LPS]) of these atypical strains using different methods and a panel of monoclonal antibodies (MAbs) directed against several epitopes of the
Brucella
O-polysaccharide (O-PS) and R-LPS. Among the most striking results,
Brucella
sp. strain BO2, isolated from a patient with chronic destructive pneumonia, showed a completely distinct S-LPS profile in silver stain gels that looked more similar to that of enterobacterial S-LPS. This strain also failed to react with MAbs against
Brucella
O-PS epitopes and showed weak reactivity with anti-R-LPS MAbs.
B. inopinata
reference strain BO1 displayed an M-dominant S-LPS type with some heterogeneity relative to the classical M-dominant
Brucella
S-LPS type. Australian wild rodent strains belonging also to the
B. inopinata
group showed a classical A-dominant S-LPS but lacked the O-PS common (C) epitopes, as previously reported for
B. suis
biovar 2 strains. Interestingly, some strains also failed to react with anti-R-LPS MAbs, such as the
B. microti
reference strain and
B. inopinata
BO1, suggesting modifications in the core-lipid A moieties of these strains. These results have several implications for serological typing and serological diagnosis and underline the need for novel tools for detection and correct identification of such novel emerging
Brucella
spp.
Schizosaccharomyces pombe Hat1 (Kat1) Is Associated with Mis16 and Is Required for Telomeric Silencing
Authors: Tong Kevin , Keller Thomas , Hoffman Charles S. , Annunziato Anthony T.
Published: July 2012

The Hat1 histone acetyltransferase has been implicated in the acetylation of histone H4 during chromatin assembly. In this study, we have characterized the Hat1 complex from the fission yeast
Schizosaccharomyces pombe
and have examined its role in telomeric silencing. Hat1 is found associated with the RbAp46 homologue Mis16, an essential protein. The Hat1 complex acetylates lysines 5 and 12 of histone H4, the sites that are acetylated in newly synthesized H4 in a wide range of eukaryotes. Deletion of
hat1
in
S. pombe
is itself sufficient to cause the loss of silencing at telomeres. This is in contrast to results obtained with an
S. cerevisiae hat1Δ
strain, which must also carry mutations of specific acetylatable lysines in the H3 tail domain for loss of telomeric silencing to occur. Notably, deletion of
hat1
from
S. pombe
resulted in an increase of acetylation of histone H4 in subtelomeric chromatin, concomitant with derepression of this region. A similar loss of telomeric silencing was also observed after growing cells in the presence of the deacetylase inhibitor trichostatin A. However, deleting
hat1
did not cause loss of silencing at centromeres or the silent mating type locus. These results point to a direct link between Hat1, H4 acetylation, and the establishment of repressed telomeric chromatin in fission yeast.
Dual RpoH Sigma Factors and Transcriptional Plasticity in a Symbiotic Bacterium
Authors: Barnett Melanie J. , Bittner Alycia N. , Toman Carol J. , Oke Valerie , Long Sharon R.
Published: July 2012

Sinorhizobium melilotican live as a soil saprophyte and can engage in a nitrogen-fixing symbiosis with plant roots. To succeed in such diverse environments, the bacteria must continually adjust gene expression. Transcriptional plasticity in eubacteria is often mediated by alternative sigma (σ) factors interacting with core RNA polymerase. TheS. melilotigenome encodes 14 of these alternative σ factors, including two putative RpoH (“heat shock”) σ factors. We used custom Affymetrix symbiosis chips to characterize the global transcriptional response ofS. melilotirpoH1,rpoH2, andrpoH1 rpoH2mutants during heat shock and stationary-phase growth. Under these conditions, expression of over 300 genes is dependent onrpoH1andrpoH2. We mapped transcript start sites of 69rpoH-dependent genes using 5′ RACE (5′ rapid amplification of cDNA ends), which allowed us to determine putative RpoH1-dependent, RpoH2-dependent, and dual-promoter (RpoH1- and RpoH2-dependent) consensus sequences that were each used to search the genome for other potential direct targets of RpoH. The inferredS. melilotiRpoH promoter consensus sequences share features ofEscherichia coliRpoH promoters but lack extended −10 motifs.
A Single Amino Acid Substitution Changes the Self-Assembly Status of a Type IV Piliation Secretin
Authors: Nickerson Nicholas N. , Abby Sophie S. , Rocha Eduardo P. C. , Chami Mohamed , Pugsley Anthony P.
Published: July 2012

Secretins form large multimeric complexes in the outer membranes of many Gram-negative bacteria, where they function as dedicated gateways that allow proteins to access the extracellular environment. Despite their overall relatedness, different secretins use different specific and general mechanisms for their targeting, assembly, and membrane insertion. We report that all tested secretins from several type II secretion systems and from the filamentous bacteriophage f1 can spontaneously multimerize and insert into liposomes in anin vitrotranscription-translation system. Phylogenetic analyses indicate that these secretins form a group distinct from the secretins of the type IV piliation and type III secretion systems, which do not autoassemblein vitro. A mutation causing a proline-to-leucine substitution allowed PilQ secretins from two different type IV piliation systems to assemblein vitro, albeit with very low efficiency, suggesting that autoassembly is an inherent property of all secretins.
Novel Listerial Glycerol Dehydrogenase- and Phosphoenolpyruvate-Dependent Dihydroxyacetone Kinase System Connected to the Pentose Phosphate Pathway
Authors: Monniot Céline , Zébré Arthur Constant , Aké Francine Moussan Désirée , Deutscher Josef , Milohanic Eliane
Published: July 2012

Several bacteria use glycerol dehydrogenase to transform glycerol into dihydroxyacetone (Dha). Dha is subsequently converted into Dha phosphate (Dha-P) by an ATP- or phosphoenolpyruvate (PEP)-dependent Dha kinase.Listeria innocuapossesses two potential PEP-dependent Dha kinases. One is encoded by 3 of the 11 genes forming the glycerol (gol) operon. This operon also containsgolD(lin0362), which codes for a new type of Dha-forming NAD+-dependent glycerol dehydrogenase. The subsequent metabolism of Dha requires its phosphorylation via the PEP:sugar phosphotransferase system components enzyme I, HPr, and EIIADha-2 (Lin0369). P∼EIIADha-2 transfers its phosphoryl group to DhaL-2, which phosphorylates Dha bound to DhaK-2. The resulting Dha-P is probably metabolized mainly via the pentose phosphate pathway, because two genes of thegoloperon encode proteins resembling transketolases and transaldolases. In addition, purified Lin0363 and Lin0364 exhibit ribose-5-P isomerase (RipB) and triosephosphate isomerase activities, respectively. The latter enzyme converts part of the Dha-P into glyceraldehyde-3-P, which, together with Dha-P, is metabolized via gluconeogenesis to form fructose-6-P. Together with another glyceraldehyde-3-P molecule, the transketolase transforms fructose-6-P into intermediates of the pentose phosphate pathway. Thegoloperon is preceded bygolR, transcribed in the opposite orientation and encoding a DeoR-type repressor. Its inactivation causes the constitutive but glucose-repressible expression of the entiregoloperon, including the last gene, encoding a pediocin immunity-like (PedB-like) protein. Its elevated level of synthesis in thegolRmutant causes slightly increased immunity against pediocin PA-1 compared to the wild-type strain or apedB-like deletion mutant.
Nucleotides Critical for the Interaction of the Streptococcus pyogenes Mga Virulence Regulator with Mga-Regulated Promoter Sequences
Authors: Hause Lara L. , McIver Kevin S.
Published: July 2012

The Mga regulator ofStreptococcus pyogenesdirectly activates the transcription of a core regulon that encodes virulence factors such as M protein (emm), C5a peptidase (scpA), and streptococcal inhibitor of complement (sic) by directly binding to a 45-bp binding site as determined by an electrophoretic mobility shift assay (EMSA) and DNase I protection. However, by comparing the nucleotide sequences of all established Mga binding sites, we found that they exhibit only 13.4% identity with no discernible symmetry. To determine the core nucleotides involved in functional Mga-DNA interactions, the M1T1 Pemm1binding site was altered and screened for nucleotides important for DNA bindingin vitroand for transcriptional activation using a plasmid-based luciferase reporterin vivo. Following this analysis, 34 nucleotides within the Pemm1binding site that had an effect on Mga binding, Mga-dependent transcriptional activation, or both were identified. Of these critical nucleotides, guanines and cytosines within the major groove were disproportionately identified clustered at the 5′ and 3′ ends of the binding site and with runs of nonessential adenines between the critical nucleotides. On the basis of these results, a Pemm1minimal binding site of 35 bp bound Mga at a level comparable to the level of binding of the larger 45-bp site. Comparison of Pemmwith directed mutagenesis performed in the M1T1 Mga-regulated PscpAand Psicpromoters, as well as methylation interference analysis of PscpA, establish that Mga binds to DNA in a promoter-specific manner.
Physical Interaction between Coat Morphogenetic Proteins SpoVID and CotE Is Necessary for Spore Encasement in Bacillus subtilis
Authors: de Francesco Melissa , Jacobs Jake Z. , Nunes Filipa , Serrano Mónica , McKenney Peter T. , Chua Ming-Hsiu , Henriques Adriano O. , Eichenberger Patrick
Published: July 2012

Endospore formation byBacillus subtilisis a complex and dynamic process. One of the major challenges of sporulation is the assembly of a protective, multilayered, proteinaceous spore coat, composed of at least 70 different proteins. Spore coat formation can be divided into two distinct stages. The first is the recruitment of proteins to the spore surface, dependent on the morphogenetic protein SpoIVA. The second step, known as encasement, involves the migration of the coat proteins around the circumference of the spore in successive waves, a process dependent on the morphogenetic protein SpoVID and the transcriptional regulation of individual coat genes. We provide genetic and biochemical evidence supporting the hypothesis that SpoVID promotes encasement of the spore by establishing direct protein-protein interactions with other coat morphogenetic proteins. It was previously demonstrated that SpoVID directly interacts with SpoIVA and the inner coat morphogenetic protein, SafA. Here, we show by yeast two-hybrid and pulldown assays that SpoVID also interacts directly with the outer coat morphogenetic protein, CotE. Furthermore, by mutational analysis, we identified a specific residue in the N-terminal domain of SpoVID that is essential for the interaction with CotE but dispensable for the interaction with SafA. We propose an updated model of coat assembly and spore encasement that incorporates several physical interactions between the principal coat morphogenetic proteins.
Multicenter Evaluation of a Sequence-Based Protocol for Subtyping Shiga Toxins and Standardizing Stx Nomenclature
Authors: Scheutz Flemming , Teel Louise D. , Beutin Lothar , Piérard Denis , Buvens Glenn , Karch Helge , Mellmann Alexander , Caprioli Alfredo , Tozzoli Rosangela , Morabito Stefano , Strockbine Nancy A. , Melton-Celsa Angela R. , Sanchez Maria , Persson Søren , O'Brien Alison D.
Published: July 2012

When Shiga toxin-producing
Escherichia coli
(STEC) strains emerged as agents of human disease, two types of toxin were identified: Shiga toxin type 1 (Stx1) (almost identical to Shiga toxin produced by
Shigella dysenteriae
type 1) and the immunologically distinct type 2 (Stx2). Subsequently, numerous STEC strains have been characterized that express toxins with variations in amino acid sequence, some of which confer unique biological properties. These variants were grouped within the Stx1 or Stx2 type and often assigned names to indicate that they were not identical in sequence or phenotype to the main Stx1 or Stx2 type. A lack of specificity or consistency in toxin nomenclature has led to much confusion in the characterization of STEC strains. Because serious outcomes of infection have been attributed to certain Stx subtypes and less so with others, we sought to better define the toxin subtypes within the main Stx1 and Stx2 types. We compared the levels of relatedness of 285 valid sequence variants of Stx1 and Stx2 and identified common sequences characteristic of each of three Stx/Stx1 and seven Stx2 subtypes. A novel, simple PCR subtyping method was developed, independently tested on a battery of 48 prototypic STEC strains, and improved at six clinical and research centers to test the reproducibility, sensitivity, and specificity of the PCR. Using a consistent schema for nomenclature of the Stx toxins and
stx
genes by phylogenetic sequence-based relatedness of the holotoxin proteins, we developed a typing approach that should obviate the need to bioassay each newly described toxin and that predicts important biological characteristics.
Phylogenetic Distribution of CTX-M- and Non-Extended-Spectrum-β-Lactamase-Producing Escherichia coli Isolates: Group B2 Isolates, Except Clone ST131, Rarely Produce CTX-M Enzymes
Authors: Brisse Sylvain , Diancourt Laure , Laouénan Cédric , Vigan Marie , Caro Valérie , Arlet Guillaume , Drieux Laurence , Leflon-Guibout Véronique , Mentré France , Jarlier Vincent , Nicolas-Chanoine Marie-Hélène ,   the Coli β Study Group
Published: July 2012

Escherichia coli
is the species most frequently associated with clinical infections by extended-spectrum-β-lactamase (ESBL)-producing isolates, with the CTX-M ESBL enzymes being predominant and found in genetically diverse
E. coli
isolates. The main objective of this study was to compare, on the basis of a case-control design, the phylogenetic diversity of 152 CTX-M-producing and 152 non-ESBL-producing clinical
E. coli
isolates. Multilocus sequence typing revealed that even though CTX-M enzymes were largely disseminated across the diversity of
E. coli
isolates, phylogenetic group B2 showed a particularly heterogeneous situation. First, clone ST131 of group B2 was strongly associated with CTX-M production (55 [79%] of 70 isolates), with CTX-M-15 being predominant. Second, the remaining members of group B2 were significantly less frequently associated with CTX-M production (9 [12%] of 75) than
E. coli
phylogenetic groups A, B1, and D (88 [55%] of 159). CTX-M-producing ST131
E. coli
isolates were significantly more frequent in patients hospitalized in geriatric wards or long-term care facilities. Besides, the non-ESBL ST131 isolates significantly more frequently showed resistance to penicillins than the non-ESBL, non-ST131 isolates did. In conclusion, the present study emphasizes the particular antimicrobial resistance and epidemiologic characteristics of clone ST131 within group B2, which could result from the higher antibiotic exposure of this clone, as it is the predominant clone of group B2 carried in the human gut.
Detection of Kudoa septempunctata 18S Ribosomal DNA in Patient Fecal Samples from Novel Food-Borne Outbreaks Caused by Consumption of Raw Olive Flounder (Paralichthys olivaceus)
Authors: Harada Tetsuya , Kawai Takao , Jinnai Michio , Ohnishi Takahiro , Sugita-Konishi Yoshiko , Kumeda Yuko
Published: July 2012

Kudoa septempunctata
is a newly identified myxosporean parasite of olive flounder (
Paralichthys olivaceus
) and a suspected causative agent of several food-borne gastroenteritis outbreaks in Japan. Here, we report the detection of
K. septempunctata
18S ribosomal DNA in fecal samples of outbreak patients using an efficient method based on real-time PCR. We first performed a spiking experiment to assess whether our previously developed real-time PCR assay was applicable to detect
K. septempunctata
in feces. Simultaneously, we compared the relative extraction efficacy of
K. septempunctata
DNA using three commercial kits. Finally, our detection method was validated by testing 45 clinical samples obtained from 13 food-borne outbreaks associated with the consumption of raw flounder and 41 fecal samples from diarrhea patients epidemiologically unrelated to the ingestion of raw fish. We found that the FastDNA Spin Kit for Soil (MP Biomedicals) was the most efficient method for extracting
K. septempunctata
DNA from fecal samples. Using this kit, the detection limit of our real-time PCR assay was 1.6 × 10
1
spores per g of feces, and positive results were obtained for 21 fecal and 2 vomitus samples obtained from the food-borne outbreaks. To our knowledge, this is the first report to describe the detection of
K. septempunctata
DNA in patient fecal samples. We anticipate that our detection method will be useful for confirming food-borne diseases caused by
K. septempunctata
in laboratory investigations.
Hepatitis C Virus-Mediated Inhibition of Cathepsin S Increases Invariant-Chain Expression on Hepatocyte Surface
Authors: Kim Hangeun , Mazumdar Budhaditya , Bose Sandip K. , Meyer Keith , Di Bisceglie Adrian M. , Hoft Daniel F. , Ray Ranjit
Published: July 2012

Hepatocytes are the main source of hepatitis C virus (HCV) replication and contain the maximum viral load in an infected person. Chronic HCV infection is characterized by weak cellular immune responses to viral proteins. Cathepsin S is a lysosomal cysteine protease and controls HLA-DR–antigen complex presentation through the degradation of the invariant chain. In this study, we examined the effect of HCV proteins on cathepsin S expression and found it to be markedly decreased in dendritic cells (DCs) exposed to HCV or in hepatocytes expressing HCV proteins. The downregulation of cathepsin S was mediated by HCV core and NS5A proteins involving inhibition of the transcription factors interferon regulatory factor 1 (IRF-1) and upstream stimulatory factor 1 (USF-1) in gamma interferon (IFN-γ)-treated hepatocytes. Inhibition of cathepsin S by HCV proteins increased cell surface expression of the invariant chain. In addition, hepatocytes stably transfected with HCV core or NS5A inhibited HLA-DR expression. Together, these results suggested that HCV has an inhibitory role on cathepsin S-mediated major histocompatibility complex (MHC) class II maturation, which may contribute to weak immunogenicity of viral antigens in chronically infected humans.
Hepatitis E Virus Quasispecies and the Outcome of Acute Hepatitis E in Solid-Organ Transplant Patients
Authors: Lhomme Sebastien , Abravanel Florence , Dubois Martine , Sandres-Saune Karine , Rostaing Lionel , Kamar Nassim , Izopet Jacques
Published: July 2012

Hepatitis E virus (HEV) infections are responsible for chronic hepatitis in immunocompromised patients, and this can evolve to cirrhosis. Like all RNA viruses, HEV exists as a mixture of heterogeneous viruses defining quasispecies. The relationship between the genetic heterogeneity described as a quasispecies, cytokine secretion, and the outcome of acute hepatitis in immunocompromised patients remains to be elucidated. We cloned and sequenced the region encoding the M and P capsid domains of HEV from eight solid-organ transplant (SOT) patients with acute HEV infection who subsequently cleared the virus and from eight SOT patients whose infection became chronic. We analyzed the cytokines and chemokines in the sera of these SOT patients by multianalyte profiling. The nucleotide sequence entropy and genetic distances were greater in patients whose infections became chronic. A lowerKa/Ksratio was associated with the persistence of HEV. The patients who developed chronic infection had lower serum concentrations of interleukin-1 (IL-1) receptor antagonist and soluble IL-2 receptor. Increased concentrations of the chemokines implicated in leukocyte recruitment to the liver were associated with persistent infection. Those patients with chronic HEV infection and progressing liver fibrosis had less quasispecies diversification during the first year than patients without liver fibrosis progression. Great quasispecies heterogeneity, a weak inflammatory response, and high serum concentrations of the chemokines involved in leukocyte recruitment to the liver in the acute phase were associated with persistent HEV infection. Slow quasispecies diversification during the first year was associated with rapidly developing liver fibrosis.
Combination of Immune and Viral Factors Distinguishes Low-Risk versus High-Risk HIV-1 Disease Progression in HLA-B*5701 Subjects
Authors: Norström Melissa M. , Buggert Marcus , Tauriainen Johanna , Hartogensis Wendy , Prosperi Mattia C. , Wallet Mark A. , Hecht Frederick M. , Salemi Marco , Karlsson Annika C.
Published: July 2012

HLA-B*5701 is the host factor most strongly associated with slow HIV-1 disease progression, although rates can vary within this group. Underlying mechanisms are not fully understood but likely involve both immunological and virological dynamics. The present study investigated HIV-1
in vivo
evolution and epitope-specific CD8
+
T cell responses in six HLA-B*5701 patients who had not received antiretroviral treatment, monitored from early infection for up to 7 years. The subjects were classified as high-risk progressors (HRPs) or low-risk progressors (LRPs) based on baseline CD4
+
T cell counts. Dynamics of HIV-1 Gag p24 evolution and multifunctional CD8
+
T cell responses were evaluated by high-resolution phylogenetic analysis and polychromatic flow cytometry, respectively. In all subjects, substitutions occurred more frequently in flanking regions than in HLA-B*5701-restricted epitopes. In LRPs, p24 sequence diversity was significantly lower; sequences exhibited a higher degree of homoplasy and more constrained mutational patterns than HRPs. The HIV-1 intrahost evolutionary rate was also lower in LRPs and followed a strict molecular clock, suggesting neutral genetic drift rather than positive selection. Additionally, polyfunctional CD8
+
T cell responses, particularly to TW10 and QW9 epitopes, were more robust in LRPs, who also showed significantly higher interleukin-2 (IL-2) production in early infection. Overall, the findings indicate that HLA-B*5701 patients with higher CD4 counts at baseline have a lower risk of HIV-1 disease progression because of the interplay between specific HLA-linked immune responses and the rate and mode of viral evolution. The study highlights the power of a multidisciplinary approach, integrating high-resolution evolutionary and immunological data, to understand mechanisms underlying HIV-1 pathogenesis.
Binding of Cellular Export Factor REF/Aly by Kaposi's Sarcoma-Associated Herpesvirus (KSHV) ORF57 Protein Is Not Required for Efficient KSHV Lytic Replication
Authors: Li Da-Jiang , Verma Dinesh , Swaminathan Sankar
Published: July 2012

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein is expressed early during lytic KSHV replication, enhances expression of many KSHV genes, and is essential for virus production. ORF57 is a member of a family of proteins conserved among all human and many animal herpesviruses that are multifunctional regulators of gene expression and act posttranscriptionally to increase accumulation of their target mRNAs. The mechanism of ORF57 action is complex and may involve effects on mRNA transcription, stability, and export. ORF57 directly binds to REF/Aly, a cellular RNA-binding protein component of the TREX complex that mediates RNA transcription and export. We analyzed the effects of an ORF57 mutation known to abrogate REF/Aly binding and demonstrate that the REF-binding mutant is impaired in activation of viral mRNAs and noncoding RNAs confined to the nucleus. Although the inability to bind REF leads to decreased ORF57 activity in enhancing gene expression, there is no demonstrable effect on nuclear export of viral mRNA or the ability of ORF57 to support KSHV replication and virus production. These data indicate that REF/Aly-ORF57 interaction is not essential for KSHV lytic replication but may contribute to target RNA stability independent of effects on RNA export, suggesting a novel role for REF/Aly in viral RNA metabolism.
N-Linked Glycosylation of GP5 of Porcine Reproductive and Respiratory Syndrome Virus Is Critically Important for Virus Replication
In Vivo
Authors: Wei Zuzhang , Lin Tao , Sun Lichang , Li Yanhua , Wang Xiaoming , Gao Fei , Liu Runxia , Chen Chunyan , Tong Guangzhi , Yuan Shishan
Published: July 2012

It has been proposed that the N-linked glycan addition at certain sites in GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) is important for production of infectious viruses and viral infectivity. However, such specific N-linked glycosylation sites do not exist in some field PRRSV isolates. This implies that the existence of GP5-associated glycan
per se
is not vital to the virus life cycle. In this study, we found that mutation of individual glycosylation sites at N30, N35, N44, and N51 in GP5 did not affect virus infectivity in cultured cells. However, the mutants carrying multiple mutations at N-linked glycosylation sites in GP5 had significantly reduced virus yields compared with the wild-type (wt) virus. As a result, no viremia and antibody response were detected in piglets that were injected with a mutant without all N-linked glycans in GP5. These results suggest that the N-linked glycosylation of GP5 is critically important for virus replication
in vivo
. The study also showed that removal of N44-linked glycan from GP5 increased the sensitivity of mutant virus to convalescent-phase serum samples but did not elicit a high-level neutralizing antibody response to wt PRRSV. The results obtained from the present study have made significant contributions to better understanding the importance of glycosylation of GP5 in the biology of PRRSV.
The Role of Clonal Interference in the Evolutionary Dynamics of Plasmid-Host Adaptation
Authors: Hughes Julie M. , Lohman Brian K. , Deckert Gail E. , Nichols Eric P. , Settles Matt , Abdo Zaid , Top Eva M.
Published: July 2012

Promiscuous plasmids replicate in a wide range of bacteria and therefore play a key role in the dissemination of various host-beneficial traits, including antibiotic resistance. Despite the medical relevance, little is known about the evolutionary dynamics through which drug resistance plasmids adapt to new hosts and thereby persist in the absence of antibiotics. We previously showed that the incompatibility group P-1 (IncP-1) minireplicon pMS0506 drastically improved its stability in novel host
Shewanella oneidensis
MR-1 after 1,000 generations under antibiotic selection for the plasmid. The only mutations found were those affecting the N terminus of the plasmid replication initiation protein TrfA1. Our aim in this study was to gain insight into the dynamics of plasmid evolution. Changes in stability and genotype frequencies of pMS0506 were monitored in evolving populations of MR-1 (pMS0506). Genotypes were determined by sequencing
trfA1
amplicons from individual clones and by 454 pyrosequencing of whole plasmids from entire populations. Stability of pMS0506 drastically improved by generation 200. Many evolved plasmid genotypes with point mutations as well as in-frame and frameshift deletions and duplications in
trfA1
were observed in all lineages with both sequencing methods. Strikingly, multiple genotypes were simultaneously present at high frequencies (>10%) in each population. Their relative abundances changed over time, but after 1,000 generations only one or two genotypes dominated the populations. This suggests that hosts with different plasmid genotypes were competing with each other, thus affecting the evolutionary trajectory. Plasmids can thus rapidly improve their stability, and clonal interference plays a significant role in plasmid-host adaptation dynamics.

IMPORTANCE
Promiscuous plasmids play an important role in the spread of antibiotic resistance and many other traits between closely and distantly related bacteria. However, little is known about the dynamics by which these broad-host-range antibiotic resistance plasmids adapt to novel bacteria and thereby become more persistent, even in the absence of antibiotics. In this study, we show that after no more than 200 generations of growth in the presence of antibiotics, a plasmid that was initially poorly maintained in a novel bacterial host evolved to become drastically more persistent in the absence of antibiotics. In each of the evolving populations, an unexpectedly large number of bacterial variants arose with distinct mutations in the plasmid’s replication initiation protein. Our results suggest that clonal interference, characterized by competition between variant clones in a population, plays a major role in the evolution of the persistence of drug resistance.