bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Analysis of MmeI from Methanol UtilizerMethylophilus methylotrophus, a Subtype IIC Restriction-Modification Enzyme Related to Type I Enzymes
Authors: Nakonieczna Joanna , Kaczorowski Tadeusz , Obarska-Kosinska Agnieszka , Bujnicki Janusz M. 
Published: November 2008

ABSTRACTMmeI fromMethylophilus methylotrophusbelongs to the type II restriction-modification enzymes. It recognizes an asymmetric DNA sequence, 5′-TCCRAC-3′ (R indicates G or A), and cuts both strands at fixed positions downstream of the specific site. This particular feature has been exploited in transcript profiling of complex genomes (using serial analysis of gene expression technology). We have shown previously that the endonucleolytic activity of MmeI is strongly dependent on the presence ofS-adenosyl-l-methionine (J. Nakonieczna, J. W. Zmijewski, B. Banecki, and A. J. Podhajska, Mol. Biotechnol. 37:127-135, 2007), which puts MmeI in subtype IIG. The same cofactor is used by MmeI as a methyl group donor for modification of an adenine in the upper strand of the recognition site toN6-methyladenine. Both enzymatic activities reside in a single polypeptide (919 amino acids [aa]), which puts MmeI also in subtype IIC of the restriction-modification systems. Based on a molecular model, generated with the use of bioinformatic tools and validated by site-directed mutagenesis, we were able to localize three functional domains in the structure of the MmeI enzyme: (i) the N-terminal portion containing the endonucleolytic domain with the catalytic Mg2+-binding motif D70-X9-EXK82, characteristic for the PD-(D/E)XK superfamily of nucleases; (ii) a central portion (aa 310 to 610) containing nine sequence motifs conserved amongN6-adenine γ-class DNA methyltransferases; (iii) the C-terminal portion (aa 610 to 919) containing a putative target recognition domain. Interestingly, all three domains showed highest similarity to the corresponding elements of type I enzymes rather than to classical type II enzymes. We have found that MmeI variants deficient in restriction activity (D70A, E80A, and K82A) can bind and methylate specific nucleotide sequence. This suggests that domains of MmeI responsible for DNA restriction and modification can act independently. Moreover, we have shown that a single amino acid residue substitution within the putative target recognition domain (S807A) resulted in a MmeI variant with a higher endonucleolytic activity than the wild-type enzyme.
Intestine and Environment of the Chicken as Reservoirs for Extraintestinal Pathogenic
Escherichia coli
Strains with Zoonotic Potential
Authors: Ewers Christa , Antão Esther-Maria , Diehl Ines , Philipp Hans-C. , Wieler Lothar H. 
Published: November 2008

ABSTRACT

Although research has increasingly focused on the pathogenesis of avian pathogenic
Escherichia coli
(APEC) infections and the “APEC pathotype” itself, little is known about the reservoirs of these bacteria. We therefore compared outbreak strains isolated from diseased chickens (
n
= 121) with nonoutbreak strains, including fecal
E. coli
strains from clinically healthy chickens (
n
= 211) and strains from their environment (
n
= 35) by determining their virulence gene profiles, phylogenetic backgrounds, responses to chicken serum, and in vivo pathogenicities in a chicken infection model. In general, by examining 46 different virulence-associated genes we were able to distinguish the three groups of avian strains, but some specific fecal and environmental isolates had a virulence gene profile that was indistinguishable from that determined for outbreak strains. In addition, a substantial number of phylogenetic EcoR group B2 strains, which are known to include potent human and animal extraintestinal pathogenic
E. coli
(ExPEC) strains, were identified among the APEC strains (44.5%) as well as among the fecal
E. coli
strains from clinically healthy chickens (23.2%). Comparably high percentages (79.2 to 89.3%) of serum-resistant strains were identified for all three groups of strains tested, bringing into question the usefulness of this phenotype as a principal marker for extraintestinal virulence. Intratracheal infection of 5-week-old chickens corroborated the pathogenicity of a number of nonoutbreak strains. Multilocus sequence typing data revealed that most strains that were virulent in chicken infection experiments belonged to sequence types that are almost exclusively associated with extraintestinal diseases not only in birds but also in humans, like septicemia, urinary tract infection, and newborn meningitis, supporting the hypothesis that not the ecohabitat but the phylogeny of
E. coli
strains determines virulence. These data provide strong evidence for an avian intestinal reservoir hypothesis which could be used to develop intestinal intervention strategies. These strains pose a zoonotic risk because either they could be transferred directly from birds to humans or they could serve as a genetic pool for ExPEC strains.
Survey of Wastewater Indicators and Human Pathogen Genomes in Biosolids Produced by Class A and Class B Stabilization Treatments
Authors: Viau Emily , Peccia Jordan 
Published: November 2008

ABSTRACTAccurate modeling of the infectious aerosol risk associated with the land application of biosolids requires an in-depth knowledge of the magnitudes and changes in pathogen concentrations for a variety of class A and class B stabilization methods. The following survey used quantitative PCR (qPCR) and culture assays to detect environmentally resistant bacterial and viral pathogens and biosolid indicator organisms for 36 biosolid grab samples. Biosolids were collected from 14 U.S. states and included 16 class B mesophilic anaerobic digestion (MAD) samples and 20 class A biosolid samples from temperature-phased anaerobic digestion (TPAD), MAD plus composting (COM), and MAD plus heat pelletization processes. The indicator concentrations of fecal coliforms and male-specific coliphages as well as pathogen genome concentrations for human adenovirus species,Legionella pneumophila,Staphylococcus aureus, andClostridium difficilewere significantly lower in the class A samples, and a multivariate analysis of variance ranked the stabilization processes from the lowest pathogen/indicator load to the highest as (i) class A COM, (ii) class A TPAD, and (iii) class B MAD. Human adenovirus genomes were found in 88% of the class B samples and 70 to 100% of the class A samples.L. pneumophila,S. aureus, andC. difficilegenomes were detected at the qPCR assay detection limits in 19 to 50% of the class B and class A anaerobic digestion samples, whileL. pneumophilawas detected in 50% of the class A compost samples. When considering all the stabilization methods, both the fecal coliform and the male-specific coliphage concentrations show a significant linear correlation with the pathogen genome concentrations. This survey provides the necessary pathogen concentrations to add to biosolid aerosol risk and pathogen exposure analyses and clarifies the effectiveness of class A stabilization methods with the pathogen and indicator loads in biosolids.
Abundance and Activity of Methanotrophic Bacteria in Littoral and Profundal Sediments of Lake Constance (Germany)
Authors: Rahalkar M. , Deutzmann J. , Schink B. , Bussmann I. 
Published: November 2008

ABSTRACT

The abundances and activities of aerobic methane-oxidizing bacteria (MOB) were compared in depth profiles of littoral and profundal sediments of Lake Constance, Germany. Abundances were determined by quantitative PCR (qPCR) targeting the
pmoA
gene and by fluorescence in situ hybridization (FISH), and data were compared to methane oxidation rates calculated from high-resolution concentration profiles. qPCR using type I MOB-specific
pmoA
primers indicated that type I MOB represented a major proportion in both sediments at all depths. FISH indicated that in both sediments, type I MOB outnumbered type II MOB at least fourfold. Results obtained with both techniques indicated that in the littoral sediment, the highest numbers of methanotrophs were found at a depth of 2 to 3 cm, corresponding to the zone of highest methane oxidation activity, although no oxygen could be detected in this zone. In the profundal sediment, highest methane oxidation activities were found at a depth of 1 to 2 cm, while MOB abundance decreased gradually with sediment depth. In both sediments, MOB were also present at high numbers in deeper sediment layers where no methane oxidation activity could be observed.
Overproduction of Exopolysaccharides by an
Escherichia coli
K-12
rpoS
Mutant in Response to Osmotic Stress
Authors: Ionescu Michael , Belkin Shimshon 
Published: November 2008

ABSTRACT

The
yjbEFGH
operon is implicated in the production of an exopolysaccharide of an unknown function and is induced by osmotic stress and negatively regulated by the general stress response sigma factor RpoS. Despite the obvious importance of RpoS, negative selection for
rpoS
has been reported to take place in starved cultures, suggesting an adaptive occurrence allowing the overexpression of RpoD-dependent uptake and nutrient-scavenging systems. The trade-off of the RpoS-dependent functions for improved nutrient utilization abilities makes the bacterium more sensitive to environmental stressors, e.g., osmotic stress. In this work, we addressed the hypothesis that overinduction of genes in
rpoS
-deficient strains indicates their essentiality. Using DNA microarrays, real-time PCR, and transcriptional fusions, we show that genes of the
wca
operon, implicated in the production of the colanic acid exopolysaccharide, previously shown to be induced by osmotic stress, are also negatively controlled by RpoS. Both exopolysaccharides in the synthesis of which
yjb
and
wca
are involved are overproduced in an
rpoS
mutant during osmotic stress. We also show that both operons are essential in an
rpoS
-deficient strain but not in the wild type; promoters of both operons are constitutively active in
yjb rpoS
mutants; this strain produces extremely mucoid colonies, forms long filaments, and exhibits a reduced growth capability. In addition, the
wca rpoS
mutant's growth is inhibited by osmotic stress. These results indicate that although induced in the wild type, both operons are much more valuable for an
rpoS
-deficient strain, suggesting that the overproduction of both exopolysaccharides is an adaptive action.
The Plasmid-Encoded Regulator Activates Factors Conferring Lysozyme Resistance on Enteropathogenic
Escherichia coli
Strains
Authors: Salinger Nina , Kokona Bashkim , Fairman Robert , Okeke Iruka N. 
Published: November 2008

ABSTRACT

We demonstrate that enhanced lysozyme resistance of enteropathogenic
Escherichia coli
requires the plasmid-encoded regulator, Per, and is mediated by factors outside the locus for enterocyte effacement. EspC, a Per-activated serine protease autotransporter protein, conferred enhanced resistance on nonpathogenic
E. coli
, and a second Per-regulated,
espC
-independent lysozyme resistance mechanism was identified.
Population Structure ofVibrio fischeriwithin the Light Organs ofEuprymna scolopesSquid from Two Oahu (Hawaii) Populations
Authors: Wollenberg M. S. , Ruby E. G. 
Published: November 2008

ABSTRACTWe resolved the intraspecific diversity ofVibrio fischeri, the bioluminescent symbiont of the Hawaiian sepiolid squidEuprymna scolopes, at two previously unexplored morphological and geographical scales. These scales ranged from submillimeter regions within the host light organ to the several kilometers encompassing two host populations around Oahu. To facilitate this effort, we employed both novel and standard genetic and phenotypic assays of light-organ symbiont populations. AV. fischeri-specific fingerprinting method and five phenotypic assays were used to gauge the genetic richness ofV. fischeripopulations; these methods confirmed that the symbiont population present in each adult host's light organ is polyclonal. Upon statistical analysis of these genetic and phenotypic population data, we concluded that the characteristics of symbiotic populations were more similar within individual host populations than between the two distinct Oahu populations ofE. scolopes, providing evidence that local geographic symbiont population structure exists. Finally, to better understand the genesis of symbiont diversity within host light organs, the process of symbiosis initiation in newly hatched juvenile squid was examined both experimentally and by mathematical modeling. We concluded that, after the juvenile hatches, only one or two cells ofV. fischerienter each of six internal epithelium-lined crypts present in the developing light organ. We hypothesize that the expansion of different, crypt-segregated, clonal populations creates the polyclonal adult light-organ population structure observed in this study. The stability of the luminous-bacterium-sepiolid squid mutualism in the presence of a polyclonal symbiont population structure is discussed in the context of contemporary evolutionary theory.
Apicoplast and Mitochondrion in Gametocytogenesis of
Plasmodium falciparum
Authors: Okamoto Noriko , Spurck Timothy P. , Goodman Christopher D. , McFadden Geoffrey I. 
Published: November 2008

ABSTRACT

Live cell imaging of human malaria parasites
Plasmodium falciparum
during gametocytogenesis revealed that the apicoplast does not grow, whereas the mitochondrion undergoes remarkable morphological development. A close connection of the two organelles is consistently maintained. The apicoplast and mitochondrion are not components of the male gametes, suggesting maternal inheritance.
Small-Molecule Type III Secretion System Inhibitors Block Assembly of the
Shigella
Type III Secreton
Authors: Veenendaal Andreas K. J. , Sundin Charlotta , Blocker Ariel J. 
Published: November 2008

ABSTRACT

Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in
Yersinia
,
Chlamydia
, and
Salmonella
species. Here we show that these are also effective on the T3SS of
Shigella flexneri
, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds.
BacA, an ABC Transporter Involved in Maintenance of Chronic Murine Infections withMycobacterium tuberculosis
Authors: Domenech Pilar , Kobayashi Hajime , LeVier Kristin , Walker Graham C. , Barry Clifton E. 
Published: November 2008

ABSTRACTBacA is an inner membrane protein associated with maintenance of chronic infections in several diverse host-pathogen interactions. To understand the function of thebacAgene inMycobacterium tuberculosis(Rv1819c), we insertionally inactivated this gene and analyzed the resulting mutant for a variety of phenotypes. BacA deficiency inM. tuberculosisdid not affect sensitivity to detergents, acidic pH, and zinc, indicating that there was no global compromise in membrane integrity, and a comprehensive evaluation of the major lipid constituents of the cell envelope failed to reveal any significant differences. Infection of mice with this mutant revealed no impact on establishment of infection but a profound effect on maintenance of extended chronic infection and ultimate outcome. As in alphaproteobacteria, deletion of BacA inM. tuberculosisled to increased bleomycin resistance, and heterologous expression of theM. tuberculosisBacA homolog inEscherichia coliconferred sensitivity to antimicrobial peptides. These results suggest a striking conservation of function for BacA-related proteins in transport of a critical molecule that determines the outcome of the host-pathogen interaction.
Difficulties of residents in training in end-of-life care. A qualitative study
Authors: Luthy C , Cedraschi C , Pautex S , Rentsch D , Piguet V , Allaz AF 
Published: November 2008

Residents in training are first-line physicians in hospital settings and they are in the process of developing knowledge and mastering clinical skills. They have to confront complex tasks calling upon their personal background, professional identity and relationships with the patients. We conducted a qualitative study investigating the difficulties they perceive in end-of-life care. In all, 24 consecutive residents were presented with a written query asking them to indicate the difficulties they identify in the management of patients hospitalised for end-of-life care. Their responses were submitted to content analysis. Physicians’ mean age was 28 ± 2.2 years, 37% were women, average postgraduate training duration was 2.5 ± 1.3 years. Content analysis elicited eight categories of difficulties: abitily to provide adequate explanations, understand the patients’ needs, have sufficient theoretical knowledge, avoid flight, avoid false reassurance, manage provision of time, face one’s limits as a physician and be able to help despite everything. Residents’ responses showed that they identify the complexity of care in terminally-ill patients early in their training. Their responses pointed to the ‘right distance’ in-between getting involved and preserving oneself as a dimension of major importance.
Crystal-chemical study of Rc natural oxides along the eskolaite – karelianite – hematite (Cr2O3–V2O3–Fe2O3) join
Authors: Secco L. , Nestola F. , Dal Negro A. , Reznitsky L. Z. 
Published: November 2008

AbstractSix natural crystals from the Sludyanka crystalline complex belonging to the eskolaite (Cr2O3)–karelianite (V2O3)–hematite (Fe2O3) solid solution were studied by means of X-ray diffraction and electron microprobe. The Fe3+-poor samples show a general increase in a and c cell parameters with increasing mean cationic radius (MCR), consistent with that shown by the synthetic crystals along the eskolaite–karelianite join. The Fe3+-richer sample deviates significantly from the behaviour shown by the Fe3+-poor ones, similar to synthetic and natural hematites; with increasing MCR, the a and c cell parameters increase linearly along the eskolaite-karelianite join. However, for the samples rich in Fe3+, from karelianite to hematite, a shows a slightly steeper slope whereas the c parameter decreases strongly. The octahedral distortion increases slightly as a function of MCR along the eskolaite-karelianite join, whereas it increases markedly for Fe3+-rich samples. The evolution of the octahedral edges and of the octahedral distortions as a function of MCR are responsible for the behaviour of the unit-cell parameters along the eskolaite-karelianite-hematite join.
Gemmological investigation of a synthetic blue beryl: a multi-methodological study
Authors: Adamo I. , Gatta G. D. , N. Rotiroti  , Diella V. , Pavese A. 
Published: November 2008

AbstractA multi-methodological investigation of a synthetic Cu/Fe-bearing blue beryl [IV(Be2.86Cu0.14)∑=3.00VI(Al1.83Fe3+0.14Mn2+0.03Mg0.03)∑=2.03IV(Si5.97Al0.03∑=6.00O18.(Li0.12Na0.04.0.40H2O)] has been performed by means of gemmological standard testing, electron microprobe chemical analyses, laser ablation inductively coupled plasma mass spectroscopy, thermo-gravimetric analyses, infrared spectroscopy and single-crystal X-ray diffraction in order to determine the gemmological properties, crystal structure and crystal-chemistry of this material. The increasing production of marketable hydrothermal synthetic beryls with 'exotic' colours and the small number of studies on the accurate location of chromophores in the crystal structure inspired this multi-methodological investigation. The X-ray structural refinements confirm that the space group of the Cu/Fe-bearing blue beryl is P6/mcc, with unit-cell parameters: 9.2483 ≤ a ≤ 9.2502 Å and 9.2184 ≤ c ≤ 9.2211 Å. The analysis of the difference Fourier maps of the electron density suggests that Cu is located at the tetrahedral site (Wyckoff 6fposition) along with Be, whereas Fe shares the octahedral site with Al (4c position). No evidence of extra-framework Cu/Fe-sites (i.e. channel sites) has been found. The Li is probably located at the extra-framework 2b site. Infrared spectra show that the H2O molecules are present with two configurations: one with the H···H vector oriented ‖[0001] and the other with H···H vector oriented ⊥[0001].
Low-temperature crystal structure evolution of (Na,Ca)(Cr,Mg)Si2O6 pyroxene
Authors: Nestola F. , Madsen A. , Tribaudino M. , Balić-Žunić T. , Ohashi H. , Secco L. , Dal Negro A. 
Published: November 2008

AbstractThe crystal structure of a clinopyroxene with composition (Na0.75Ca0.25)(Cr0.75Mg0.25)Si2O6 was refined at 100, 150, 200, 250 and 298 K. The work was performed in the context of an investigation on the low-temperature behaviour of A+M3+Si2O6 (with A dominant in Na and M = transition elements) pyroxenes in order to provide new insights concerning the phase transition and anomalies recently found in the low-temperature behaviour of NaTiSi2O6 and NaGaSi2O6 compounds. The refinements were done in the C2/c space group (wR2 between 0.048 and 0.068), and no change of symmetry was observed down to 100 K. Highly-anisotropic axial thermal expansion occurs with the scheme αb ≥ αb > αc.The M2, M1 and T polyhedra expand with αM2 > αM1 αT as generally observed in pyroxenes. A discontinuity in the M1 polyhedral volume is observed between 200 and 250 K, similar to the one observed in NaGaSi2O6 between 190 and 235 K.The atomic displacement parameters are scaled according to the following pattern: UM2 > UO2 > UO3 ≥ U01 > UT ≥ UM1 Comparison with previous data along the CaMgSi2O-NaCrSi2O6 join suggests significant positional disorder for the O1 oxygen, due to repulsion of the 2p orbitals of O1 and the non-bondine 3d electrons of Cr.