bluuebunny/crossref_metadata_2025 huggingface dataset:
Fabrication of gold tips suitable for tip-enhanced Raman spectroscopy
Authors: Williams Craig , Roy Debdulal
Published: October 2008

The authors report on a simple method of fabricating gold tips, suitable for use in a shear-force mode atomic force microscope for tip-enhanced Raman spectroscopy (TERS). The proposed electrochemical method is highly optimized and offers five advantages: produces a low-aspect ratio tip that can withstand the lateral force at the end of the tip during scanning, produces a sharp tip end to generate good Raman enhancements for TERS, a self-terminating process that makes the etching process easy, use of inexpensive electronic systems so that it can be adopted by any laboratory, and less time consuming (takes less than 20s to fabricate a single tip). This article demonstrates TERS imaging using such tips.
Roughness effects on contact angle measurements
Authors: Ryan Bernard J. , Poduska Kristin M.
Published: October 2008

We have developed a simple and economical procedure to demonstrate the effects of roughness and wetting fraction on the equilibrium contact angles of liquid droplets on solid surfaces. Contact angles for droplets placed on a rough surface, which wet only a portion of the surface, are larger than the contact angles of droplets formed by condensation of steam, which wet the surface more completely. These contact angle data facilitate assessments of changes in true surface area, due to surface roughening, as well as changes in the fractional contact areas of the water droplets, due to the formation of air pockets between the rough surface and the droplet.
Quantum noise detection: A portable and educational system
Authors: Morizur Jean-François , Colla Massimiliano , Bachor Hans-A.
Published: October 2008

Quantum noise is a key feature of laser beams. It is both a limiting effect in contemporary optical measurements and a manifestation of the quantum nature of light. Its properties distinguish it from classical noise. We demonstrate a simple, reliable, and portable apparatus using low cost commercial lasers and electronics that provides evidence of these properties.
Genotypic Resistance Analysis of the Virological Response to Fosamprenavir-Ritonavir in Protease Inhibitor-Experienced Patients in CONTEXT and TRIAD Clinical Trials
Authors: Marcelin Anne-Geneviève , Flandre Philippe , Molina Jean-Michel , Katlama Christine , Yeni Patrick , Raffi Francois , Antoun Zeina , Ait-Khaled Mounir , Calvez Vincent
Published: October 2008

The aim of this study was to identify human immunodeficiency virus (HIV) protease mutations associated with virological response (VR) to fosamprenavir-ritonavir (FPV/r) in 113 protease inhibitor (PI)-experienced patients randomized in both CONTEXT and TRIAD clinical trials and receiving the same dose (700/100 mg twice daily) of FPV/r. The impact of each protease mutation on the VR to FPV/r, defined as the decrease in HIV RNA at week 12, was investigated with nonparametric analyses. A step-by-step procedure was done using a Jonckheere-Terpstra (JT) test that retains the group of mutations most strongly associated with the VR. Mutations at the following 14 codons were associated with a reduced VR to FPV/r: 10, 15, 33, 46, 54, 60, 62, 63, 72, 73, 82, 84, 89, and 90. The JT procedure led to selecting the CONTEXT/TRIAD genotypic set of mutations, I15V, M46I/L, I54L/M/V, D60E, L63P/T, and I84V, as providing the strongest association with the VR (P= 1.45 × 10−11). In the nine patients with zero mutations within this set, the median decrease in HIV RNA was −2.63 log copies/ml, and was −2.22 (n= 45), −1.50 (n= 26), −0.58 (n= 23), −0.47 (n= 6), −0.13 (n= 3), and 0.04 (n= 1) log copies/ml in those with one, two, three, four, five, and six mutations, respectively. This study identified six mutations associated with VR to FPV/r. Some of these mutations are shared with the current FPV/r Agence Nationale de Recherches sur le SIDA (ANRS) resistance score, which has been cross-validated in the CONTEXT/TRIAD data set, suggesting that the current ANRS FPV/r score is a useful tool for the prediction of VR to FPV/r in PI-experienced patients.
Characterization of pUO-StVR2, a Virulence-Resistance Plasmid Evolved from the pSLT Virulence Plasmid of
Salmonella enterica
Serovar Typhimurium
Authors: Herrero Ana , Mendoza M. Carmen , Rodicio Rosaura , Rodicio M. Rosario
Published: October 2008

pUO-StVR2 is a virulence-resistance plasmid which originated from pSLT of
Salmonella enterica
serovar Typhimurium through acquisition of a complex resistance island, flanked by regions that provide a toxin-antitoxin system and an iron uptake system. The presence of resistance and virulence determinants on the same plasmid allows coselection of both properties, potentially increasing health risks.
Chloroquine Resistance-Conferring Mutations in
pfcrt
Give Rise to a Chloroquine-Associated H
+
Leak from the Malaria Parasite's Digestive Vacuole
Authors: Lehane Adele M. , Kirk Kiaran
Published: October 2008

Chloroquine resistance in the malaria parasite
Plasmodium falciparum
is conferred by mutations in the
P. falciparum c
hloroquine
r
esistance
t
ransporter (PfCRT). PfCRT localizes to the membrane of the parasite's internal digestive vacuole, an acidic organelle in which chloroquine accumulates to high concentrations and exerts its toxic effect. Mutations in PfCRT are thought to reduce chloroquine accumulation in this organelle. How they do so is the subject of ongoing debate. Recently we have shown that in the presence of chloroquine there is an increased leak of H
+
from the digestive vacuole in chloroquine-resistant but not chloroquine-sensitive parasites. Here, using transfectant parasite strains of a single genetic background and differing only in their
pfcrt
allele, we show that chloroquine resistance-conferring PfCRT mutations are responsible for this chloroquine-associated H
+
leak. This is consistent with the hypothesis that the chloroquine resistance-conferring forms of PfCRT mediate the efflux of chloroquine, in association with H
+
, from the malaria parasite's digestive vacuole.
Pharmacodynamics of Cidofovir for Vaccinia Virus Infection in an In Vitro Hollow-Fiber Infection Model System
Authors: McSharry James J. , Deziel Mark R. , Zager Kris , Weng Qingmei , Drusano George L.
Published: October 2008

Variola major virus
remains a potent weapon of bioterror. There is currently an investigational-new-drug application for cidofovir for the therapy of variola major virus infections. Stittelaar and colleagues compared the levels of effectiveness of postexposure smallpox vaccination (Elstree-RIVM) and antiviral treatment with cidofovir or an acyclic nucleoside phosphonate analogue {6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidine (HPMPO-DAPy)} after lethal intratracheal infection of cynomolgus monkeys with monkeypox virus, a variola virus surrogate. Their results demonstrated that either compound was more effective than vaccination with the Ellstree vaccine (K. J. Stittelaar et al., Nature 439:745-748, 2006). An unanswered question is how to translate this information into therapy for poxvirus infections in people. In a proof-of-principle study, we used a novel in vitro hollow-fiber infection model system to determine the pharmacodynamics of vaccinia virus infection of HeLa-S3 cells treated with cidofovir. Our results demonstrate that the currently licensed dose of cidofovir of 5 mg/kg of body weight weekly with probenecid (which ameliorates nephrotoxicity) is unlikely to provide protection for patients intentionally exposed to
Variola major virus
. We further demonstrate that the antiviral effect is independent of the schedule of drug administration. Exposures (area under the concentration-time curve) to cidofovir that will have a robust protective effect will require doses that are 5 to 10 times that currently administered to humans. Such doses may cause nephrotoxicity, and therefore, approaches that include probenecid administration as well as schedules of administration that will help ameliorate the uptake of cidofovir into renal tubular epithelial cells need to be considered when addressing such treatment for people.
Identification of Potent Bactericidal Compounds Produced by Escapin, an
l
-Amino Acid Oxidase in the Ink of the Sea Hare
Aplysia californica
Authors: Ko Ko-Chun , Wang Binghe , Tai Phang C. , Derby Charles D.
Published: October 2008

The ink of sea hares (
Aplysia californica
) contains escapin, an
l
-amino acid oxidase that metabolizes
l
-lysine, thereby producing a mixture that kills microbes and deters attacking predators. This secretion contains H
2
O
2
, ammonia, and an equilibrium mixture of “escapin intermediate product” (EIP-K) that includes α-keto-ε-aminocaproic acid and several other molecules. Components of the equilibrium mixture react nonenzymatically with H
2
O
2
to form “escapin end product” (EEP-K), which contains δ-aminovaleric acid and δ-valerolactam. The proportions of the molecules in this equilibrium mixture change with pH, and this is biologically important because the secretion is pH 5 when released but becomes pH 8 when fully diluted in seawater. The goal of the current study was to identify which molecules in this equilibrium mixture are bactericidal. We show that a mixture of H
2
O
2
and EIP-K, but not EEP-K, at low mM concentrations is synergistically responsible for most of the bactericidal activity of the secretion against
Escherichia coli
,
Vibrio harveyi
,
Staphylococcus aureus
, and
Pseudomonas aeruginosa
. Low pH enhances the bactericidal effect, and this does not result from stress associated with low pH itself. Sequential exposure to low mM concentrations of EIP-K and H
2
O
2
, in either order, does not kill
E. coli
. Reaction products formed when
l
-arginine is substituted for
l
-lysine have almost no bactericidal activity. Our results favor the idea that the bactericidal activity is due to unstable intermediates of the reaction of α-keto-ε-aminocaproic acid with H
2
O
2
.
Cross-Kingdom Amplification Using
Bacteria
-Specific Primers: Complications for Studies of Coral Microbial Ecology
Authors: Galkiewicz Julia P. , Kellogg Christina A.
Published: October 2008

PCR amplification of pure bacterial DNA is vital to the study of bacterial interactions with corals. Commonly used
Bacteria
-specific primers 8F and 27F paired with the universal primer 1492R amplify both eukaryotic and prokaryotic rRNA genes. An alternative primer set, 63F/1542R, is suggested to resolve this problem.
Novel Cold-Adapted Alkaline Lipase from an Intertidal Flat Metagenome and Proposal for a New Family of Bacterial Lipases
Authors: Kim Eun-Young , Oh Ki-Hoon , Lee Mi-Hwa , Kang Chul-Hyung , Oh Tae-Kwang , Yoon Jung-Hoon
Published: October 2008

A new lipase, LipEH166, isolated from an intertidal flat metagenome, showed no amino acid similarity to any known lipolytic enzyme except in the consensus region. This suggested that LipEH166 and its homologues belong to a new family of lipolytic enzymes. Partial characterization indicated that LipEH166 is a novel cold-adapted alkaline lipase.
Adenoviruses in Immunocompromised Hosts
Authors: Echavarría Marcela
Published: October 2008

The number of patients with acquired immunodeficiency has grown steadily as a result of both a larger number of patients receiving solid organ and hematopoietic stem cell transplants and their longer survival times. The use of newer, more potent immunosuppressive regimens has increased the frequency of severe adenovirus infections. Human adenoviruses are a large group of viruses, represented by at least 52 serotypes with various genotypes divided into genomic clusters, and these may cause a broad variety of clinical manifestations. The development of molecular methods has increased the sensitivity and rapidity of adenovirus infection diagnosis. The implementation of PCR assays has significantly contributed to the identification of patients with disseminated adenovirus disease. More recently, the development of real-time PCR assays has permitted virus quantification and patient follow-up. There is no treatment for adenovirus with demonstrated efficacy, although cidofovir is widely used. Sensitive diagnostic tests for adenovirus can contribute to the early diagnosis and successful treatment of life-threatening adenovirus infections, especially in complex immunocompromised patients. The development of improved adenovirus therapy still remains a challenge. Adenovirus genetic diversity should be considered for diagnosis, typing, and therapeutic interventions.
A
Phytophthora sojae
G-Protein α Subunit Is Involved in Chemotaxis to Soybean Isoflavones
Authors: Hua Chenlei , Wang Yonglin , Zheng Xiaobo , Dou Daolong , Zhang Zhengguang , Govers Francine , Wang Yuanchao
Published: October 2008

For the soybean pathogen
Phytophthora sojae
, chemotaxis of zoospores to isoflavones is believed to be critical for recognition of the host and for initiating infection. However, the molecular mechanisms underlying this chemotaxis are largely unknown. To investigate the role of G-protein and calcium signaling in chemotaxis, we analyzed the expression of several genes known to be involved in these pathways and selected one that was specifically expressed in sporangia and zoospores but not in mycelium. This gene, named
PsGPA1
, is a single-copy gene in
P. sojae
and encodes a G-protein α subunit that shares 96% identity in amino acid sequence with that of
Phytophthora infestans
. To elucidate the function, expression of
PsGPA1
was silenced by introducing antisense constructs into
P. sojae. PsGPA1
silencing did not disturb hyphal growth or sporulation but severely affected zoospore behavior, including chemotaxis to the soybean isoflavone daidzein. Zoospore encystment and cyst germination were also altered, resulting in the inability of the
PsGPA1
-silenced mutants to infect soybean. In addition, the expressions of a calmodulin gene,
PsCAM1
, and two calcium- and calmodulin-dependent protein kinase genes,
PsCMK3
and
PsCMK4
, were increased in the mutant zoospores, suggesting that
PsGPA1
negatively regulates the calcium signaling pathways that are likely involved in zoospore chemotaxis.
Functional Characterization of AasP, a Maturation Protease Autotransporter Protein ofActinobacillus pleuropneumoniae
Authors: Ali Tehmeena , Oldfield Neil J. , Wooldridge Karl G. , Turner David P. , Ala'Aldeen Dlawer A. A.
Published: October 2008

Actinobacillus pleuropneumoniaeis the etiological agent of porcine pleuropneumonia, a highly contagious respiratory infection in pigs. AasP, a putative subtilisin-like serine protease autotransporter, has recently been identified inA. pleuropneumoniae. We hypothesized that, similarly to other autotransporters of this type, AasP may undergo autocatalytic cleavage resulting in release of the passenger domain of the protein. Furthermore, AasP may be responsible for cleavage of otherA. pleuropneumoniaeouter membrane proteins. To address these hypotheses, theaasPgene was cloned and the expressed recombinant AasP protein used to raise monospecific rabbit antiserum. Immunoblot analysis of whole-cell lysates and secreted proteins demonstrated that AasP does not undergo proteolytic cleavage. Immunoblot analysis also confirmed that AasP is universally expressed byA. pleuropneumoniae. Confirmation of the maturation protease function of AasP was obtained through phenotypic analysis of anA. pleuropneumoniae aasPdeletion mutant and by functional complementation. Comparison of the secreted proteins of the wild type, anaasPmutant derivative, and anaasPmutant complemented intransled to the identification of OmlA protein fragments that were present only in the secreted-protein preparations of the wild-type and complemented strains, indicating that AasP is involved in modification of OmlA. This is the first demonstration of a function for any autotransporter protein inActinobacillus pleuropneumoniae.
Mode of Expression and Functional Characterization of FCT-3 Pilus Region-Encoded Proteins in
Streptococcus pyogenes
Serotype M49
Authors: Nakata Masanobu , Köller Thomas , Moritz Karin , Ribardo Deborah , Jonas Ludwig , McIver Kevin S. , Sumitomo Tomoko , Terao Yutaka , Kawabata Shigetada , Podbielski Andreas , Kreikemeyer Bernd
Published: October 2008

The human pathogen
Streptococcus pyogenes
(group A streptococcus [GAS]) pilus components, suggested to play a role in pathogenesis, are encoded in the variable FCT (fibronectin- and collagen-binding T-antigen) region. We investigated the functions of sortase A (SrtA), sortase C2 (SrtC2), and the FctA protein of the most prevalent type 3 FCT region from a serotype M49 strain. Although it is considered a housekeeping sortase, SrtA's activity is involved in pilus formation in addition to its essentiality for GAS extracellular matrix protein binding, host cell adherence/internalization, survival in human blood, and biofilm formation. SrtC2 activity is crucial for pilus formation but dispensable for the other phenotypes tested in vitro. FctA is the major pilus backbone protein, simultaneously acting as the M49 T antigen, and requires SrtC2 and LepA, a signal peptidase I homologue, for monomeric surface expression and polymerization, respectively. Collagen-binding protein Cpa expression supports pilus formation at the pilus base. Immunofluorescence microscopy and fluorescence-activated cell sorting analysis revealed several unexpected expression patterns, as follows: (i) the monomeric pilus protein FctA was found exclusively at the old poles of GAS cells, (ii) FctA protein expression increased with lower temperatures, and (iii) FctA protein expression was restricted to 20 to 50% of a given GAS M49 population, suggesting regulation by a bistability mode. Notably, disruption of pilus assembly by sortase deletion rendered GAS serotype M49 significantly more aggressive in a dermonecrotic mouse infection model, indicating that sortase activity and, consequently, pilus expression allow a subpopulation of this GAS serotype to be less aggressive. Thus, pilus expression may not be a virulence attribute of GAS per se.
Staphylococcus aureus
Elicits Marked Alterations in the Airway Proteome during Early Pneumonia
Authors: Ventura Christy L. , Higdon Roger , Hohmann Laura , Martin Daniel , Kolker Eugene , Liggitt H. Denny , Skerrett Shawn J. , Rubens Craig E.
Published: October 2008

Pneumonia caused by
Staphylococcus aureus
is a growing concern in the health care community. We hypothesized that characterization of the early innate immune response to bacteria in the lungs would provide insight into the mechanisms used by the host to protect itself from infection. An adult mouse model of
Staphylococcus aureus
pneumonia was utilized to define the early events in the innate immune response and to assess the changes in the airway proteome during the first 6 h of pneumonia.
S. aureus
actively replicated in the lungs of mice inoculated intranasally under anesthesia to cause significant morbidity and mortality. By 6 h postinoculation, the release of proinflammatory cytokines caused effective recruitment of neutrophils to the airway. Neutrophil influx, loss of alveolar architecture, and consolidated pneumonia were observed histologically 6 h postinoculation. Bronchoalveolar lavage fluids from mice inoculated with phosphate-buffered saline (PBS) or
S. aureus
were depleted of overabundant proteins and subjected to strong cation exchange fractionation followed by liquid chromatography and tandem mass spectrometry to identify the proteins present in the airway. No significant changes in response to PBS inoculation or 30 min following
S. aureus
inoculation were observed. However, a dramatic increase in extracellular proteins was observed 6 h postinoculation with
S. aureus
, with the increase dominated by inflammatory and coagulation proteins. The data presented here provide a comprehensive evaluation of the rapid and vigorous innate immune response mounted in the host airway during the earliest stages of
S. aureus
pneumonia.
Role for the Chlamydial Type III Secretion Apparatus in Host Cytokine Expression
Authors: Prantner Daniel , Nagarajan Uma M.
Published: October 2008

In many important human pathogens, such asShigellaandSalmonellaspp., the bacterial type III secretion (T3S) apparatus is required to initiate inflammation via activation of caspase-1- or NF-κB-dependent genes. Using an ex vivo infection model, the goal of the present study was to determine whether the chlamydial T3S apparatus also modulates the host inflammatory response. Infections of mouse peritoneal macrophages were performed withChlamydia muridarum, and the expression of inflammatory cytokines was monitored by quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay. Since there is no current genetic system forChlamydiaspp., blockade of T3S was accomplished pharmacologically using a T3S inhibitor called INP0007. It has been previously shown that INP0007 also blocks chlamydial growth in vitro and that the addition of exogenous iron completely reverses this deficit. The addition of iron to INP0007-treatedC. muridarum-infected macrophages not only restored chlamydial growth deficit caused by INP0007 but also led to a multi-inclusion phenotype. Overall, T3S inhibition led to decreased interleukin-6 (IL-6), IL-1β, and CXCL10, whereas the tumor necrosis factor alpha levels were unchanged. Rescue of chlamydial growth by addition of iron sulfate did not restore cytokine production, implying that the decreased expression of many cytokines during infection was dependent on T3S and not solely on growth. In addition, the observation that the greatest effects of INP0007 were seen at late time points during infection suggests that a temporally regulated T3S effector protein(s) may be triggering the host cytokine response.
Factor H-Binding Protein Is Important for Meningococcal Survival in Human Whole Blood and Serum and in the Presence of the Antimicrobial Peptide LL-37
Authors: Seib K. L. , Serruto D. , Oriente F. , Delany I. , Adu-Bobie J. , Veggi D. , Aricò B. , Rappuoli R. , Pizza M.
Published: October 2008

Factor H-binding protein (fHBP; GNA1870) is one of the antigens of the recombinant vaccine against serogroup B
Neisseria meningitidis
, which has been developed using reverse vaccinology and is the basis of a meningococcal B vaccine entering phase III clinical trials. Binding of factor H (fH), an inhibitor of the complement alternative pathway, to fHBP enables
N. meningitidis
to evade killing by the innate immune system. All fHBP null mutant strains analyzed were sensitive to killing in ex vivo human whole blood and serum models of meningococcal bacteremia with respect to the isogenic wild-type strains. The fHBP mutant strains of MC58 and BZ83 (high fHBP expressors) survived in human blood and serum for less than 60 min (decrease of >2 log
10
CFU), while NZ98/254 (intermediate fHBP expressor) and 67/00 (low fHBP expressor) showed decreases of >1 log
10
CFU after 60 to 120 min of incubation. In addition, fHBP is important for survival in the presence of the antimicrobial peptide LL-37 (decrease of >3 log
10
CFU after 2 h of incubation), most likely due to electrostatic interactions between fHBP and the cationic LL-37 molecule. Hence, the expression of fHBP by
N. meningitidis
strains is important for survival in human blood and human serum and in the presence of LL-37, even at low levels. The functional significance of fHBP in mediating resistance to the human immune response, in addition to its widespread distribution and its ability to induce bactericidal antibodies, indicates that it is an important component of the serogroup B meningococcal vaccine.
Pathogenic Potential of Emergent Sorbitol-Fermenting
Escherichia coli
O157:NM
Authors: Rosser Tracy , Dransfield Tracy , Allison Lesley , Hanson Mary , Holden Nicola , Evans Judith , Naylor Stuart , La Ragione Roberto , Low J. Christopher , Gally David L.
Published: October 2008

Non-sorbitol-fermenting (NSF)
Escherichia coli
O157:H7 is the primary Shiga toxin-producing
E. coli
(STEC) serotype associated with human infection. Since 1988, sorbitol-fermenting (SF) STEC O157:NM strains have emerged and have been associated with a higher incidence of progression to hemolytic-uremic syndrome (HUS) than NSF STEC O157:H7. This study investigated bacterial factors that may account for the increased pathogenic potential of SF STEC O157:NM. While no evidence of toxin or toxin expression differences between the two O157 groups was found, the SF STEC O157:NM strains adhered at significantly higher levels to a human colonic cell line. Under the conditions tested, curli were shown to be the main factor responsible for the increased adherence to Caco-2 cells. Notably, 52 of 66 (79%) European SF STEC O157:NM strains tested bound Congo red at 37
ο
C and this correlated with curli expression. In a subset of strains, curli expression was due to increased expression from the
csgBAC
promoter that was not always a consequence of increased
csgD
expression. The capacity of SF STEC O157:NM strains to express curli at 37
ο
C may have relevance to the epidemiology of human infections as curliated strains could promote higher levels of colonization and inflammation in the human intestine. In turn, this could lead to increased toxin exposure and an increased likelihood of progression to HUS.
SMC Protein-Dependent Chromosome Condensation during Aerial Hyphal Development in
Streptomyces
Authors: Kois Agnieszka , Świątek Magdalena , Jakimowicz Dagmara , Zakrzewska-Czerwińska Jolanta
Published: October 2008

Members of the SMC (
s
tructural
m
aintenance of
c
hromosomes) protein family play a central role in higher-order chromosome dynamics from bacteria to humans. So far, studies of bacterial SMC proteins have focused only on unicellular rod-shaped organisms that divide by binary fission. The conversion of multigenomic aerial hyphae of the mycelial organism
Streptomyces coelicolor
into chains of unigenomic spores requires the synchronous segregation of multiple chromosomes. Here we focus on the contribution of SMC proteins to sporulation-associated chromosome segregation in
S. coelicolor
. Deletion of the
smc
gene causes aberrant DNA condensation and missegregation of chromosomes (7.5% anucleate spores). In vegetative mycelium, immunostained SMC proteins were observed sporadically, while in aerial hyphae about to undergo sporulation they appeared as irregularly spaced foci which accompanied but did not colocalize with ParB complexes. Our data demonstrate that efficient chromosome segregation requires the joint action of SMC and ParB proteins. SMC proteins, similarly to ParAB and FtsZ, presumably belong to a larger group of proteins whose expression is highly induced in response to the requirement of aerial hyphal maturation.
A Novel Thermostable Arylesterase from the ArchaeonSulfolobus solfataricusP1: Purification, Characterization, and Expression
Authors: Park Young-Jun , Yoon Sung-Jin , Lee Hee-Bong
Published: October 2008

A novel thermostable arylesterase, a 35-kDa monomeric enzyme, was purified from the thermoacidophilic archaeonSulfolobus solfataricusP1. The optimum temperature and pH were 94°C and 7.0, respectively. The enzyme displayed remarkable thermostability: it retained 52% of its activity after 50 h of incubation at 90°C. In addition, the purified enzyme showed high stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity besides showing an arylesterase activity toward aromatic esters: it exhibits not only carboxylesterase activity toward tributyrin andp-nitrophenyl esters containing unsubstituted fatty acids from butyrate (C4) to palmitate (C16), but also paraoxonase activity toward organophosphates such asp-nitrophenylphosphate, paraoxon, and methylparaoxon. Thekcat/Kmratios of the enzyme for phenyl acetate and paraoxon, the two most preferable substrates among all tested, were 30.6 and 119.4 s−1·μM−1, respectively. The arylesterase gene consists of 918 bp corresponding to 306 amino acid residues. The deduced amino acid sequence shares 34% identity with that of arylesterase fromAcinetobactersp. strain ADP1. Furthermore, we successfully expressed active recombinantS. solfataricusarylesterase inEscherichia coli. Together, our results show that the enzyme is a serine esterase belonging to the A-esterases and contains a catalytic triad composed of Ser156, Asp251, and His281 in the active site.
Comparative Genome Sequence Analysis of Multidrug-Resistant
Acinetobacter baumannii
Authors: Adams Mark D. , Goglin Karrie , Molyneaux Neil , Hujer Kristine M. , Lavender Heather , Jamison Jennifer J. , MacDonald Ian J. , Martin Kristienna M. , Russo Thomas , Campagnari Anthony A. , Hujer Andrea M. , Bonomo Robert A. , Gill Steven R.
Published: October 2008

The recent emergence of multidrug resistance (MDR) in
Acinetobacter baumannii
has raised concern in health care settings worldwide. In order to understand the repertoire of resistance determinants and their organization and origins, we compared the genome sequences of three MDR and three drug-susceptible
A. baumannii
isolates. The entire MDR phenotype can be explained by the acquisition of discrete resistance determinants distributed throughout the genome. A comparison of closely related MDR and drug-susceptible isolates suggests that drug efflux may be a less significant contributor to resistance to certain classes of antibiotics than inactivation enzymes are. A resistance island with a variable composition of resistance determinants interspersed with transposons, integrons, and other mobile genetic elements is a significant but not universal contributor to the MDR phenotype. Four hundred seventy-five genes are shared among all six clinical isolates but absent from the related environmental species
Acinetobacter baylyi
ADP1. These genes are enriched for transcription factors and transporters and suggest physiological features of
A. baumannii
that are related to adaptation for growth in association with humans.
Biosynthesis of 7-Deazaguanosine-Modified tRNA Nucleosides: a New Role for GTP Cyclohydrolase I
Authors: Phillips Gabriella , El Yacoubi Basma , Lyons Benjamin , Alvarez Sophie , Iwata-Reuyl Dirk , de Crécy-Lagard Valérie
Published: October 2008

Queuosine (Q) and archaeosine (G
+
) are hypermodified ribonucleosides found in tRNA. Q is present in the anticodon region of tRNA
GUN
in
Eukarya
and
Bacteria
, while G
+
is found at position 15 in the D-loop of archaeal tRNA. Prokaryotes produce these 7-deazaguanosine derivatives de novo from GTP through the 7-cyano-7-deazaguanine (pre-Q
0
) intermediate, but mammals import the free base, queuine, obtained from the diet or the intestinal flora. By combining the results of comparative genomic analysis with those of genetic studies, we show that the first enzyme of the folate pathway, GTP cyclohydrolase I (GCYH-I), encoded in
Escherichia coli
by
folE
, is also the first enzyme of pre-Q
0
biosynthesis in both prokaryotic kingdoms. Indeed, tRNA extracted from an
E. coli
Δ
folE
strain is devoid of Q and the deficiency is complemented by expressing GCYH-I-encoding genes from different bacterial or archaeal origins. In a similar fashion, tRNA extracted from a
Haloferax volcanii
strain carrying a deletion of the GCYH-I-encoding gene contains only traces of G
+
. These results link the production of a tRNA-modified base to primary metabolism and further clarify the biosynthetic pathway for these complex modified nucleosides.
Influence of thePseudomonasQuinolone Signal on Denitrification inPseudomonas aeruginosa
Authors: Toyofuku Masanori , Nomura Nobuhiko , Kuno Eriko , Tashiro Yosuke , Nakajima Toshiaki , Uchiyama Hiroo
Published: October 2008

Denitrification is a well-studied respiratory system that is also important in the biogeochemical nitrogen cycle. Environmental signals such as oxygen and N-oxides have been demonstrated to regulate denitrification, though how denitrification is regulated in a bacterial community remains obscure.Pseudomonas aeruginosais a ubiquitous bacterium that controls numerous genes through cell-to-cell signals. The bacterium possesses at least twoN-acyl-l-homoserine lactone (AHL) signals. In our previous study, these quorum-sensing signals controlled denitrification inP. aeruginosa. In addition to the AHL signals, a third cell-to-cell communication signal, 2-heptyl-3-hydroxy-4-quinolone, referred to as thePseudomonasquinolone signal (PQS), has been characterized. In this study, we examined the effect of PQS on denitrification to obtain more insight into the respiratory regulation in a bacterial community. Denitrification inP. aeruginosawas repressed by PQS, which was partially mediated by PqsR and PqsE. Measuring the denitrifying enzyme activities indicated that nitrite reductase activity was increased by PQS, whereas PQS inhibited nitric oxide reductase and the nitrate-respiratory chain activities. This is the first report to demonstrate that PQS influences enzyme activities, suggesting this effect is not specific toP. aeruginosa. Furthermore, when iron was supplied to the PQS-added medium, denitrifying activity was almost restored, indicating that the iron chelating property of PQS affected denitrification. Thus, our data indicate that PQS regulates denitrification primarily through iron chelation. The PQS effect on denitrification was relevant in a condition where oxygen was limited and denitrification was induced, suggesting its role in controlling denitrification where oxygen is present.
Different Signaling Roles of Two Conserved Residues in the Cytoplasmic Hairpin Tip of Tsr, the
Escherichia coli
Serine Chemoreceptor
Authors: Mowery Patricia , Ostler Jeffery B. , Parkinson John S.
Published: October 2008

Bacterial chemoreceptors form ternary signaling complexes with the histidine kinase CheA through the coupling protein CheW. Receptor complexes in turn cluster into cellular arrays that produce highly sensitive responses to chemical stimuli. In
Escherichia coli
, receptors of different types form mixed trimer-of-dimers signaling teams through the tips of their highly conserved cytoplasmic domains. To explore the possibility that the hairpin loop at the tip of the trimer contact region might promote interactions with CheA or CheW, we constructed and characterized mutant receptors with amino acid replacements at the two nearly invariant hairpin charged residues of Tsr: R388, the most tip-proximal trimer contact residue, and E391, the apex residue of the hairpin turn. Mutant receptors were subjected to in vivo tests for the assembly and function of trimers, ternary complexes, and clusters. All R388 replacements impaired or destroyed Tsr function, apparently through changes in trimer stability or geometry. Large-residue replacements locked R388 mutant ternary complexes in the kinase-off (F, H) or kinase-on (W, Y) signaling state, suggesting that R388 contributes to signaling-related conformational changes in the trimer. In contrast, most E391 mutants retained function and all formed ternary signaling complexes efficiently. Hydrophobic replacements of any size (G, A, P, V, I, L, F, W) caused a novel phenotype in which the mutant receptors produced rapid switching between kinase-on and -off states, indicating that hairpin tip flexibility plays an important role in signal state transitions. These findings demonstrate that the receptor determinants for CheA and CheW binding probably lie outside the hairpin tip of the receptor signaling domain.
Metabolic Network Model of a Human Oral Pathogen
Authors: Mazumdar Varun , Snitkin Evan S. , Amar Salomon , Segrè Daniel
Published: October 2008

The microbial community present in the human mouth is engaged in a complex network of diverse metabolic activities. In addition to serving as energy and building-block sources, metabolites are key players in interspecies and host-pathogen interactions. Metabolites are also implicated in triggering the local inflammatory response, which can affect systemic conditions such as atherosclerosis, obesity, and diabetes. While the genome of several oral pathogens has been sequenced, quantitative understanding of the metabolic functions of any oral pathogen at the system level has not been explored yet. Here we pursue the computational construction and analysis of the genome-scale metabolic network ofPorphyromonas gingivalis, a gram-negative anaerobe that is endemic in the human population and largely responsible for adult periodontitis. Integrating information from the genome, online databases, and literature screening, we built a stoichiometric model that encompasses 679 metabolic reactions. By using flux balance approaches and automated network visualization, we analyze the growth capacity under amino-acid-rich medium and provide evidence that amino acid preference and cytotoxic by-product secretion rates are suitably reproduced by the model. To provide further insight into the basic metabolic functions ofP. gingivalisand suggest potential drug targets, we study systematically how the network responds to any reaction knockout. We focus specifically on the lipopolysaccharide biosynthesis pathway and identify eight putative targets, one of which has been recently verified experimentally. The current model, which is amenable to further experimental testing and refinements, could prove useful in evaluating the oral microbiome dynamics and in the development of novel biomedical applications.
Identification of Two Genes, sll0804 and slr1306, as Putative Components of the CO
2
-Concentrating Mechanism in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Zhang Shulu , Spann Kevin W. , Frankel Laurie K. , Moroney James V. , Bricker Terry M.
Published: October 2008

Insertional transposon mutations in the sll0804 and slr1306 genes were found to lead to a loss of optimal photoautotrophy in the cyanobacterium
Synechocystis
sp. strain PCC 6803 grown under ambient CO
2
concentrations (350 ppm). Mutants containing these insertions (4BA2 and 3ZA12, respectively) could grow photoheterotrophically on glucose or photoautotrophically at elevated CO
2
concentrations (50,000 ppm). Both of these mutants exhibited an impaired affinity for inorganic carbon. Consequently, the Sll0804 and Slr1306 proteins appear to be putative components of the carbon-concentrating mechanism in
Synechocystis
sp. strain PCC 6803.
Molecular detection of nematicidal crystalliferousBacillus thuringiensisstrains of Iran and evaluation of their toxicity on free-living and plant-parasitic nematodes
Authors: Salehi Jouzani Gholamreza , Seifinejad Ali , Saeedizadeh Abbas , Nazarian Amin , Yousefloo Majid , Soheilivand Saeed , Mousivand Maryam , Jahangiri Rosa , Yazdani Mehdi , Amiri Reza Maali , Akbari Sepideh
Published: October 2008

The characterization of nematode-effective strains and cry genes in the Iranian Bacillus thuringiensis (Bt) collection (70 isolates) is presented. Characterization was based on PCR analysis using 12 specific primers for cry5, cry6, cry12, cry13, cry14, and cry21 genes encoding proteins active against nematodes, crystal morphology, and protein band patterns as well as their nematicidal activity on root-knot nematode ( Meloidogyne incognita ) and two free-living nematodes ( Chiloplacus tenuis and Acrobeloides enoplus ). PCR results with primers for these genes showed that 22 isolates (31.5%) contain a minimum of one nematode-active cry gene. Strains containing the cry6 gene were the most abundant and represent 22.8% of the isolates. Bt strains harboring cry14 genes were also abundant (14.2%). cry21 and cry5 genes were less abundant, found in 4.2% and 2.8% of the strains, respectively. In total, six different nematode-active cry gene profiles were detected in this collection. Four isolates did not show the expected PCR product size for cry5, cry6, and cry21 genes; they might contain potentially novel insecticidal crystal protein genes. Twenty-two Bt isolates containing nematode-active cry genes were selected for preliminary bioassays on M. incognita. Based on these bioassays, four isolates were selected for detailed bioassays. Isolates YD5 and KON4 at 2 × 108 CFU/mL concentrations showed 77% and 81% toxicity on M. incognita, respectively. The free-living nematodes C. tenuis and A. enoplus were more susceptible and the highest mortality was observed within 48 h of incubation at all of the concentrations tested. Maximum mortality was recorded for isolates SN1 and KON4 at 2 × 108 CFU/mL concentrations and resulted in 68% and 77% adults deaths of C. tenuis and 68% and 72% for A. enoplus, respectively. Our results showed that PCR is a useful technique for toxicity prediction of nematicidal Bt isolates.
Enhanced plant nutrient use efficiency with PGPR and AMF in an integrated nutrient management system
Authors: Adesemoye A.O. , Torbert H.A. , Kloepper J.W.
Published: October 2008

A 3 year field study was conducted with field corn from 2005 to 2007 to test the hypothesis that microbial inoculants that increase plant growth and yield can enhance nutrient uptake, and thereby remove more nutrients, especially N, P, and K from the field as part of an integrated nutrient management system. The field trial evaluated microbial inoculants, which include a commercially available plant growth-promoting rhizobacteria (PGPR), arbuscular mycorrhiza fungi (AMF), and their combination across 2 tillage systems (no-till and conventional till) and 2 fertilization regimes (poultry litter and ammonium nitrate). Data were collected on plant height, yield (dry mass of ears and silage), and nutrient content of corn grain and silage. In addition, nutrient content of soil was determined, and bioavailability of soil nutrient was measured with plant root simulator probes. Results showed that inoculants promoted plant growth and yield. For example, grain yields (kg·ha–1) in 2007 for inoculants were 7717 for AMF, 7260 for PGPR+AMF, 7313 for PGPR, 5725 for the control group, and for fertilizer were 7470 for poultry litter and 6537 for NH4NO3. Nitrogen content per gram of grain tissues was significantly enhanced in 2006 by inoculant, fertilizer, and their interactions. Significantly higher amounts of N, P, and K were removed from the plots with inoculants, based on total nutrient content of grain per plot. These results supported the overall hypothesis and indicate that application of inoculants can lead to reduction in the build up of N, P, and K in agricultural soils. Further studies should be conducted to combine microbial inoculants with reduced rates of fertilizer.
Seasonal free amino acid fluctuations in red pine and white spruce needles
Authors: Kim Y.T. , Glerum. C.
Published: October 2008

Eighteen free amino acids were quantified biweekly from the needles of 3-year-old bare-root red pine (Pinusresinosa Ait.) and white spruce (Piceaglauca (Moench) Voss) seedlings over a 3-year period. In both species, concentrations of alanine, serine, aspartic acid, glutamic acid, asparagine, threonine, valine, glutamine, and proline increased during summer. Phenylalanine, lysine, and leucine accumulated in red pine during the summer but not in white spruce. During the winter tyrosine and histidine accumulated in white spruce but not in red pine. Only tryptophan consistently exhibited a significant trend in both species, rising from a low concentration in summer to a peak in fall and then returning to a low concentration in the spring. Thirteen amino acids in red pine and 15 in white spruce exhibited significant coefficients of determination with photoperiod and daily air temperature together. Fourteen amino acids in red pine and 10 in white spruce had significant photoperiod partial regression coefficients, while three amino acids in red pine and six in white spruce exhibited significant temperature partial regression coefficients. Total amino acid concentration exhibited significant coefficients of determination, with only the photoperiod partial regression coefficients being significant for both species.
Comparative analysis of the genetic composition of canopy and Juvenile sugar mapie individuals (Acersaccharum) in an old-growth forest in southern Quebec as related to anthropogenic disturbance
Authors: Simon Jean-Pierre , Payette Yves , Longpré Marie-Hélène
Published: October 2008

The unexpected discovery of a relatively undisturbed presettlement forest in southern Quebec, the Muir Forest, allowed us to investigate the potential reproductive effects of recent man-made air pollution upon the genetic structure and variability of sugar maple (Acersaccharum Marsh.) at this site. The genetic structure of the canopy subpopulation consisting of 62 trees over 140 years of age was compared with that of three understory subpopulations of 90 juvenile individuals, 6 to 15 years old. The genetic structure and variability were analyzed using cellulose acetate electrophoresis of 14 enzyme systems. The analysis of 20 loci showed an average polymorphism of 60.1%, a mean of 1.83 alleles per locus, a mean of 1.17 effective alleles, and observed and expected levels of heterozygosity of 0.136 and 0.148, respectively. There were no significant differences among the canopy and the three juvenile subpopulations for these five genetic parameters. Estimated Fis and Fit (Wright's fixation indices) values indicated, respectively, a 7.3% and a 8.7% average excess of homozygotes. There was a tendency for rare alleles at several loci to be at lower frequencies of absent in some or all of the juvenile subpopulations. On average, a higher value of Fis was obtained for the combined juvenile subpopulation than for the canopy subpopulation, but this difference was statistically not significant. The average genetic differentiation among subpopulations was weak (Fst = 0.015), and only 0.4% of the variability could be attributed to differences between the canopy and the combined juvenile subpopulations. Genetic distances between the canopy and juvenile subpopulations were very low (e.g., Nei's unbiased genetic distance = 0.000–0.003) and not different from those calculated among juvenile subpopulations. This study does not support the hypothesis that the reduction.in reproductive potential resulting from environmental pollution and from other recent anthropogenic disturbances may involve a differential gametophytic selection strong enough to reduce the genetic variability and (or) modify the genetic structure of progenies generated at the Muir Forest during the last 2 decades.
Precommercial thinning in Pseudotsuga, Tsuga, and Abies stands affected by armillaria root disease: 10-year results
Authors: Filip Gregory M. , Goheen Donald J.
Published: October 2008

Four 10- to 20-year-old stands were precommercially thinned to determine the effects of thinning on tree growth and mortality caused by armillaria root disease in the Cascade Range of western Oregon and Washington, U.S.A.: one stand of Douglas-fir (Pseudotsugamenziesii (Mirb.) Franco var. menziesii) and noble fir (Abiesprocera Rehd.), one of Douglas-fir and western hemlock (Tsugaheterophylla (Raf.) Sarg.), one of Douglas-fir alone, and one of Shasta red fir (AbiesmagnificaA. Mutt. var. shastensis Lemm.) and mountain hemlock (Tsugamertensiana (Bong.) Carr.). After 10 years, differences in crop-tree mortality between thinned and unthinned plots were not significant in any of the four stands. Tree radial growth was significantly increased by thinning in 6 of 15 plots. Crop-tree basal area (per hectare) growth was significantly greater in thinned plots. Basal area (per hectare) growth of all trees was significantly greater in unthinned plots. Apparently, from a root-disease perspective, precommercial thinning does not affect the incidence of crop-tree mortality after 10 years, but tree growth increases significantly.