bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiplex Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Quantification in Human Plasma of Fluconazole, Itraconazole, Hydroxyitraconazole, Posaconazole, Voriconazole, Voriconazole-
N
-Oxide, Anidulafungin, and Caspofungin
Authors: Decosterd Laurent Arthur , Rochat Bertrand , Pesse Benoît , Mercier Thomas , Tissot Frédéric , Widmer Nicolas , Bille Jacques , Calandra Thierry , Zanolari Boris , Marchetti Oscar
Published: September 2010

Therapeutic drug monitoring (TDM) may contribute to optimizing the efficacy and safety of antifungal therapy because of the large variability in drug pharmacokinetics. Rapid, sensitive, and selective laboratory methods are needed for efficient TDM. Quantification of several antifungals in a single analytical run may best fulfill these requirements. We therefore developed a multiplex ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method requiring 100 μl of plasma for simultaneous quantification within 7 min of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-
N
-oxide, caspofungin, and anidulafungin. Protein precipitation with acetonitrile was used in a single extraction procedure for eight analytes. After reverse-phase chromatographic separation, antifungals were quantified by electrospray ionization-triple-quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. Deuterated isotopic compounds of azole antifungals were used as internal standards. The method was validated based on FDA recommendations, including assessment of extraction yields, matrix effect variability (<9.2%), and analytical recovery (80.1 to 107%). The method is sensitive (lower limits of azole quantification, 0.01 to 0.1 μg/ml; those of echinocandin quantification, 0.06 to 0.1 μg/ml), accurate (intra- and interassay biases of −9.9 to +5% and −4.0 to +8.8%, respectively), and precise (intra- and interassay coefficients of variation of 1.2 to 11.1% and 1.2 to 8.9%, respectively) over clinical concentration ranges (upper limits of quantification, 5 to 50 μg/ml). Thus, we developed a simple, rapid, and robust multiplex UPLC-MS/MS assay for simultaneous quantification of plasma concentrations of six antifungals and two metabolites. This offers, by optimized and cost-effective lab resource utilization, an efficient tool for daily routine TDM aimed at maximizing the real-time efficacy and safety of different recommended single-drug antifungal regimens and combination salvage therapies, as well as a tool for clinical research.
LmrS Is a Multidrug Efflux Pump of the Major Facilitator Superfamily from
Staphylococcus aureus
Authors: Floyd Jody L. , Smith Kenneth P. , Kumar Sanath H. , Floyd Jared T. , Varela Manuel F.
Published: September 2010

A multidrug efflux pump designated LmrS (lincomycin resistance protein of
Staphylococcus aureus
), belonging to the major facilitator superfamily (MFS) of transporters, was cloned, and the role of LmrS in antimicrobial efflux was evaluated. The highest relative increase in MIC, 16-fold, was observed for linezolid and tetraphenylphosphonium chloride (TPCL), followed by an 8-fold increase for sodium dodecyl sulfate (SDS), trimethoprim, and chloramphenicol. LmrS has 14 predicted membrane-spanning domains and is homologous to putative lincomycin resistance proteins of
Bacillus
spp.,
Lactobacillus
spp., and
Listeria
spp.
Gene Therapeutic Approach for Inhibiting Hepatitis C Virus Replication Using a Recombinant Protein That Controls Interferon Expression
Authors: Joo Chul Hyun , Lee Uk , Nam Young Ran , Jung Jae U. , Lee Heuiran , Cho Young Keol , Kim Yoo Kyum
Published: September 2010

The hepatitis C virus (HCV) is a continuing threat to public health. The systemic administration of interferon alpha with ribavirin is the only currently approved treatment. However, this treatment is associated with a wide spectrum of systemic side effects that limits its effectiveness; thus, there is an urgent need for new treatment modalities. In this study, we describe a novel anti-HCV strategy employing a recombinant transcription factor that we have engineered in such a way that NS3/4a viral protease controls its intracellular localization, thereby restoring interferon secretion specifically in cells infected with HCV. Proof-of-concept experiments validated the strategy, showing that the recombinant transcription factor was triggered to stimulate interferon promoter by NS3/4A and remained inactive in cells without NS3/4a. Using an adenovirus-associated viral vector delivery system, we found that the recombinant transcription factor inhibited HCV replication effectively
in vitro
in cultured cells.
Colistin Resistance in
Acinetobacter baumannii
Is Mediated by Complete Loss of Lipopolysaccharide Production
Authors: Moffatt Jennifer H. , Harper Marina , Harrison Paul , Hale John D. F. , Vinogradov Evgeny , Seemann Torsten , Henry Rebekah , Crane Bethany , St. Michael Frank , Cox Andrew D. , Adler Ben , Nation Roger L. , Li Jian , Boyce John D.
Published: September 2010

Infections caused by multidrug-resistant (MDR) Gram-negative bacteria represent a major global health problem. Polymyxin antibiotics such as colistin have resurfaced as effective last-resort antimicrobials for use against MDR Gram-negative pathogens, including
Acinetobacter baumannii
. Here we show that
A. baumannii
can rapidly develop resistance to polymyxin antibiotics by complete loss of the initial binding target, the lipid A component of lipopolysaccharide (LPS), which has long been considered to be essential for the viability of Gram-negative bacteria. We characterized 13 independent colistin-resistant derivatives of
A. baumannii
type strain ATCC 19606 and showed that all contained mutations within one of the first three genes of the lipid A biosynthesis pathway:
lpxA
,
lpxC
, and
lpxD
. All of these mutations resulted in the complete loss of LPS production. Furthermore, we showed that loss of LPS occurs in a colistin-resistant clinical isolate of
A. baumannii
. This is the first report of a spontaneously occurring, lipopolysaccharide-deficient, Gram-negative bacterium.
Effect of Host Cells on Low- and Medium-Pressure UV Inactivation of Adenoviruses
Authors: Guo Huiling , Chu Xiaona , Hu Jiangyong
Published: September 2010

UV disinfection is highly effective against most pathogens, with the exception of the adenoviruses (AD). To date, many studies have focused on low-pressure (LP) UV inactivation of AD, but little is known about the effect of medium-pressure (MP) UV inactivation of AD. Despite numerous studies of LP UV inactivation of AD, extreme variabilities in the LP UV dose requirements of AD had been observed because of differing experimental conditions used, such as the types of cell lines used for AD enumeration. This study therefore investigates the effect of three different host cell lines (PLC/PRF/5, human embryonic kidney 293 [HEK293], and XP17BE) on the LP and MP UV dose requirements of AD serotype 5 (AD5), AD40, and AD41 under similar experimental settings. Results showed that for 4-log inactivation of AD, LP UV and MP UV doses needed to be in the ranges of 123 to 182 mJ/cm
2
and 65 to 90 mJ/cm
2
, respectively, when HEK293 and PLC/PRF/5 cells were used for enumeration. The UV doses required for MP UV inactivation of AD were significantly lower than those required for LP UV inactivation (
P
value < 0.05). When different cell lines were used for enumeration, UV dose requirements for AD differed. AD were portrayed to be most susceptible to UV (LP UV doses of <57 mJ/cm
2
and MP UV doses of <42 mJ/cm
2
for 4-log AD inactivation) when the XP17BE cells were used as the host cell. The use of different cell lines for AD enumeration affected LP UV dose results more significantly than MP UV dose results (
P
value < 0.05). Cell line variability factors for LP UV disinfection (CL
LP
) and MP UV disinfection (CL
MP
) for AD5, AD40, and AD41 enumerated with HEK293, PLC/PRF/5, and XP17BE cells were in the ranges of 1.0 to 3.2 and 1.0 to 2.5, respectively.
Changes in Ultrastructure and Fourier Transform Infrared Spectrum of
Salmonella enterica
Serovar Typhimurium Cells after Exposure to Stress Conditions
Authors: Alvarez-Ordóñez A. , Prieto M.
Published: September 2010

The effect of exposure to acid (pH 2.5), alkaline (pH 11.0), heat (55°C), and oxidative (40 mM H
2
O
2
) lethal conditions on the ultrastructure and global chemical composition of
Salmonella enterica
serovar Typhimurium CECT 443 cells was studied using transmission electron microscopy and Fourier transform infrared spectroscopy (FT-IR) combined with multivariate statistical methods (hierarchical cluster analysis and factor analysis). Infrared spectra exhibited marked differences in the five spectral regions for all conditions tested compared to those of nontreated control cells, which suggests the existence of a complex bacterial stress response in which modifications in a wide variety of cellular compounds are involved. The visible spectral changes observed in all of the spectral regions, together with ultrastructural changes observed by transmission electron microscopy and data obtained from membrane integrity tests, indicate the existence of membrane damage or alterations in membrane composition after heat, acid, alkaline, and oxidative treatments. Results obtained in this study indicate the potential of FT-IR spectroscopy to discriminate between intact and injured bacterial cells and between treatment technologies, and they show the adequacy of this technique to study the molecular aspects of bacterial stress response.
Dispersion of TiO
2
Nanoparticle Agglomerates by
Pseudomonas aeruginosa
Authors: Horst Allison M. , Neal Andrea C. , Mielke Randall E. , Sislian Patrick R. , Suh Won Hyuk , Mädler Lutz , Stucky Galen D. , Holden Patricia A.
Published: September 2010

Engineered nanoparticles are increasingly incorporated into consumer products and are emerging as potential environmental contaminants. Upon environmental release, nanoparticles could inhibit bacterial processes, as evidenced by laboratory studies. Less is known regarding bacterial alteration of nanoparticles, including whether bacteria affect physical agglomeration states controlling nanoparticle settling and bioavailability. Here, the effects of an environmental strain of
Pseudomonas aeruginosa
on TiO
2
nanoparticle agglomerates formed in aqueous media are described. Environmental scanning electron microscopy and cryogenic scanning electron microscopy visually demonstrated bacterial dispersion of large agglomerates formed in cell culture medium and in marsh water. For experiments in cell culture medium, quantitative image analysis verified that the degrees of conversion of large agglomerates into small nanoparticle-cell combinations were similar for 12-h-growth and short-term cell contact experiments. Dispersion in cell growth medium was further characterized by size fractionation: for agglomerated TiO
2
suspensions in the absence of cells, 81% by mass was retained on a 5-μm-pore-size filter, compared to only 24% retained for biotic treatments. Filtrate cell and agglomerate sizes were characterized by dynamic light scattering, revealing that the average bacterial cell size increased from 1.4 μm to 1.9 μm because of nano-TiO
2
biosorption. High-magnification scanning electron micrographs showed that
P. aeruginosa
dispersed TiO
2
agglomerates by preferential biosorption of nanoparticles onto cell surfaces. These results suggest a novel role for bacteria in the environmental transport of engineered nanoparticles, i.e., growth-independent, bacterially mediated size and mass alterations of TiO
2
nanoparticle agglomerates.
The Spatial Factor, Rather than Elevated CO
2
, Controls the Soil Bacterial Community in a Temperate Forest Ecosystem
Authors: Ge Yuan , Chen Chengrong , Xu Zhihong , Oren Ram , He Ji-Zheng
Published: September 2010

The global atmospheric carbon dioxide (CO
2
) concentration is expected to increase continuously over the next century. However, little is known about the responses of soil bacterial communities to elevated CO
2
in terrestrial ecosystems. This study aimed to partition the relative influences of CO
2
, nitrogen (N), and the spatial factor (different sampling plots) on soil bacterial communities at the free-air CO
2
enrichment research site in Duke Forest, North Carolina, by two independent techniques: an entirely sequencing-based approach and denaturing gradient gel electrophoresis. Multivariate regression tree analysis demonstrated that the spatial factor could explain more than 70% of the variation in soil bacterial diversity and 20% of the variation in community structure, while CO
2
or N treatment explains less than 3% of the variation. For the effects of soil environmental heterogeneity, the diversity estimates were distinguished mainly by the total soil N and C/N ratio. Bacterial diversity estimates were positively correlated with total soil N and negatively correlated with C/N ratio. There was no correlation between the overall bacterial community structures and the soil properties investigated. This study contributes to the information about the effects of elevated CO
2
and soil fertility on soil bacterial communities and the environmental factors shaping the distribution patterns of bacterial community diversity and structure in temperate forest soils.
Persistence of
Bacteroides
Species Populations in a River as Measured by Molecular and Culture Techniques
Authors: Ballesté Elisenda , Blanch Anicet R.
Published: September 2010

Given the interest in
Bacteroides
species as microbial source tracking (MST) markers, and the limited knowledge of the survival of
Bacteroides
species in the environment, here we examine the survival of
Bacteroides fragilis
,
B. thetaiotaomicron
, and environmental species of
Bacteroides
by use of culture techniques and molecular tools. Two kinds of experiments were performed: (i) on-site experiments, in which bacteria were exposed to changes in the levels of several environmental parameters in a river, and (ii) microcosm assays in the laboratory, with controlled temperatures. On-site experiments showed different survival patterns for the cultivable
Bacteroides
strains.
B. fragilis
die-off rate was strongly affected by the combined effect of high temperatures and grazing predators, which were more active under warmer conditions. However, the survival rates of cultivable
B. thetaiotaomicron
and environmental
Bacteroides
spp. were more affected by dissolved oxygen (DO) concentration in water. Environmental
Bacteroides
strains survived longer than either type strain, due to better adaptation to environmental conditions. However, the period of their survival was shorter than that observed for fecal coliforms and enterococci, suggesting
Bacteroides
species as markers of recent fecal pollution. The total
Bacteroides
species were detected by molecular techniques throughout the experiment in winter, but they were detected on only two or three days in the summer. This indicates that temperature is the main factor affecting DNA degradation, regardless of species. The use of microcosms in the laboratory also pointed to temperature as the main factor affecting
Bacteroides
survival, regardless of species. However, the conditions in the laboratory may mask the effects of the environmental factors and their interactions. The observed variability in die-off rate as a function of the species analyzed, the experimental conditions, and the methodology used should be taken into consideration in future persistence studies.
Detection of Protozoan Hosts for
Legionella pneumophila
in Engineered Water Systems by Using a Biofilm Batch Test
Authors: Valster Rinske M. , Wullings Bart A. , van der Kooij Dick
Published: September 2010

Legionella pneumophila
proliferates in aquatic habitats within free-living protozoa, 17 species of which have been identified as hosts by using
in vitro
experiments. The present study aimed at identifying protozoan hosts for
L. pneumophila
by using a biofilm batch test (BBT). Samples (600 ml) collected from 21 engineered freshwater systems, with added polyethylene cylinders to promote biofilm formation, were inoculated with
L. pneumophila
and subsequently incubated at 37°C for 20 days. Growth of
L. pneumophila
was observed in 16 of 18 water types when the host protozoan
Hartmannella vermiformis
was added. Twelve of the tested water types supported growth of
L. pneumophila
or indigenous
Legionella anisa
without added
H. vermiformis
. In 12 of 19 BBT flasks
H. vermiformis
was indicated as a host, based on the ratio between maximum concentrations of
L. pneumophila
and
H. vermiformis
, determined with quantitative PCR (Q-PCR), and the composition of clone libraries of partial 18S rRNA gene fragments. Analyses of 609 eukaryotic clones from the BBTs revealed that 68 operational taxonomic units (OTUs) showed the highest similarity to free-living protozoa. Forty percent of the sequences clustering with protozoa showed ≥99.5% similarity to
H. vermiformis
. None of the other protozoa serving as hosts in
in vitro
studies were detected in the BBTs. In several tests with growth of
L. pneumophila
, the protozoa
Diphylleia rotans
,
Echinamoeba thermarum
, and
Neoparamoeba
sp. were identified as candidate hosts.
In vitro
studies are needed to confirm their role as hosts for
L. pneumophila
. Unidentified protozoa were implicated as hosts for uncultured
Legionella
spp. grown in BBT flasks at 15°C.
Functionality of Sortase A in
Lactococcus lactis
Authors: Dieye Yakhya , Oxaran Virginie , Ledue-Clier Florence , Alkhalaf Walid , Buist Girbe , Juillard Vincent , Lee Chang Won , Piard Jean-Christophe
Published: September 2010

Lactococcus lactis
IL1403 harbors a putative sortase A (SrtA) and 11 putative sortase substrates that carry the canonical LPXTG signature of such substrates. We report here on the functionality of SrtA to anchor five LPXTG substrates to the cell wall, thus suggesting that SrtA is the housekeeping sortase in
L. lactis
IL1403.
Health Risk from the Use of Roof-Harvested Rainwater in Southeast Queensland, Australia, as Potable or Nonpotable Water, Determined Using Quantitative Microbial Risk Assessment
Authors: Ahmed W. , Vieritz A. , Goonetilleke A. , Gardner T.
Published: September 2010

A total of 214 rainwater samples from 82 tanks were collected in urban Southeast Queensland (SEQ) in Australia and analyzed for the presence and numbers of zoonotic bacterial and protozoal pathogens using binary PCR and quantitative PCR (qPCR). Quantitative microbial risk assessment (QMRA) analysis was used to quantify the risk of infection associated with the exposure to potential pathogens from roof-harvested rainwater used as potable or nonpotable water. Of the 214 samples tested, 10.7%, 9.8%, 5.6%, and 0.4% were positive for the
Salmonella invA
,
Giardia lamblia
β-giardin,
Legionella pneumophila mip
, and
Campylobacter jejuni mapA
genes, respectively.
Cryptosporidium parvum
oocyst wall protein (COWP) could not be detected. The estimated numbers of
Salmonella
,
G. lamblia
, and
L. pneumophila
organisms ranged from 6.5 × 10
1
to 3.8 × 10
2
cells, 0.6 × 10° to 3.6 × 10° cysts, and 6.0 × 10
1
to 1.7 × 10
2
cells per 1,000 ml of water, respectively. Six risk scenarios were considered for exposure to
Salmonella
spp.,
G. lamblia
, and
L. pneumophila
. For
Salmonella
spp. and
G. lamblia
, these scenarios were (i) liquid ingestion due to drinking of rainwater on a daily basis, (ii) accidental liquid ingestion due to hosing twice a week, (iii) aerosol ingestion due to showering on a daily basis, and (iv) aerosol ingestion due to hosing twice a week. For
L. pneumophila
, these scenarios were (i) aerosol inhalation due to showering on a daily basis and (ii) aerosol inhalation due to hosing twice a week. The risk of infection from
Salmonella
spp.,
G. lamblia
, and
L. pneumophila
associated with the use of rainwater for showering and garden hosing was calculated to be well below the threshold value of one extra infection per 10,000 persons per year in urban SEQ. However, the risk of infection from ingesting
Salmonella
spp. and
G. lamblia
via drinking exceeded this threshold value and indicated that if undisinfected rainwater is ingested by drinking, then the incidences of the gastrointestinal diseases salmonellosis and giardiasis are expected to range from 9.8 × 10° to 5.4 × 10
1
(with a mean of 1.2 × 10
1
from Monte Carlo analysis) and from 1.0 × 10
1
to 6.5 × 10
1
cases (with a mean of 1.6 × 10
1
from Monte Carlo analysis) per 10,000 persons per year, respectively, in urban SEQ. Since this health risk seems higher than that expected from the reported incidences of gastroenteritis, the assumptions used to estimate these infection risks are critically examined. Nonetheless, it would seem prudent to disinfect rainwater for use as potable water.
The
Rhodococcus opacus
PD630 Heparin-Binding Hemagglutinin Homolog TadA Mediates Lipid Body Formation
Authors: MacEachran Daniel P. , Prophete M. E. , Sinskey A. J.
Published: September 2010

Generally, prokaryotes store carbon as polyhydroxyalkanoate, starch, or glycogen. The Gram-positive actinomycete
Rhodococcus opacus
strain PD630 is noteworthy in that it stores carbon in the form of triacylglycerol (TAG). Several studies have demonstrated that
R. opacus
PD630 can accumulate up to 76% of its cell dry weight as TAG when grown under nitrogen-limiting conditions. While this process is well studied, the underlying molecular and biochemical mechanisms leading to TAG biosynthesis and subsequent storage are poorly understood. We designed a high-throughput genetic screening to identify genes and their products required for TAG biosynthesis and storage in
R. opacus
PD630. We identified a gene predicted to encode a putative heparin-binding hemagglutinin homolog, which we have termed
tadA
(
t
riacylglycerol
a
ccumulation
d
eficient), as being important for TAG accumulation. Kinetic studies of TAG accumulation in both the wild-type (WT) and mutant strains demonstrated that the
tadA
mutant accumulates 30 to 40% less TAG than the parental strain (WT). We observed that lipid bodies formed by the mutant strain were of a different size and shape than those of the WT. Characterization of TadA demonstrated that the protein is capable of binding heparin and of agglutinating purified lipid bodies. Finally, we observed that the TadA protein localizes to lipid bodies in
R. opacus
PD630 both
in vivo
and
in vitro
. Based on these data, we hypothesize that the TadA protein acts to aggregate small lipid bodies, found in cells during early stages of lipid storage, into larger lipid bodies and thus plays a key role in lipid body maturation in
R. opacus
PD630.
Cell Wall Anchoring of the 37-Kilodalton Oncofetal Antigen by
Lactobacillus plantarum
for Mucosal Cancer Vaccine Delivery
Authors: Fredriksen Lasse , Mathiesen Geir , Sioud Mouldy , Eijsink Vincent G. H.
Published: September 2010

The 37-kDa oncofetal antigen (OFA), a tumor immunogen expressed on all mammalian cancers examined to date, was secreted and anchored to the cell wall of
Lactobacillus plantarum
using homologous signal peptides and LPxTG anchors. Orally administered
L. plantarum
expressing anchored OFA induced a specific immune response against OFA in mice.
Use of Cellular CRISPR (Clusters of Regularly Interspaced Short Palindromic Repeats) Spacer-Based Microarrays for Detection of Viruses in Environmental Samples
Authors: Snyder Jamie C. , Bateson Mary M. , Lavin Matthew , Young Mark J.
Published: September 2010

It is currently difficult to detect unknown viruses in any given environment. The recent discovery of CRISPR (clusters of regularly interspaced short palindromic repeats) loci within bacterial and archaeal cellular genomes may provide an alternative approach to detect new viruses. It has been shown that the spacer sequences between the direct repeat units of the CRISPR loci are often derived from viruses and likely function as guide sequences to protect the cell from viral infection. The spacer sequences within the CRISPR loci may therefore serve as a record of the viruses that have replicated within the cell. We have cataloged the CRISPR spacer sequences from cellular metagenomic data from high-temperature (>80°C), acidic (pH < 4) hot spring environments located in Yellowstone National Park (YNP). We designed a microarray platform utilizing these CRISPR spacer sequences as potential probes to detect viruses present in YNP hot spring environments. We show that this microarray approach can detect viral sequences directly from virus-enriched environmental samples, detecting new viruses which have not been previously characterized. We further demonstrated that this microarray approach can be used to examine temporal changes in viral populations within the environment. Our results demonstrate that CRISPR spacer sequence-based microarrays will be useful tools for detecting and monitoring viruses from diverse environmental samples.
Salmonella entericain Commercial Swine Feed and Subsequent Isolation of Phenotypically and Genotypically Related Strains from Fecal Samples
Authors: Molla Bayleyegn , Sterman Allyson , Mathews Jennifer , Artuso-Ponte Valeria , Abley Melanie , Farmer William , Rajala-Schultz Päivi , Morrow W. E. Morgan , Gebreyes Wondwossen A.
Published: September 2010

The purpose of this study was to determine the occurrence and genotypic relatedness ofSalmonella entericaisolates recovered from feed and fecal samples in commercial swine production units. Of 275 feed samples,Salmonellawas detected in 10 feed samples that originated from 8 of 36 (22.2%) barns, with a prevalence of 3.6% (10/275 samples). In fecal samples, a prevalence of 17.2% was found at the early finishing stage (1,180/6,880 samples), with a significant reduction in prevalence (7.4%) when pigs reached market age (392/5,321 samples). Of the 280Salmonellaisolates systematically selected for further characterization, 50% of the feed isolates and 55.3% of the isolates of fecal origin showed similar phenotypes based on antimicrobial resistance patterns and serogrouping. About 44% of the isolates were multidrug resistant. Pulsed-field gel electrophoresis (PFGE) genotyping grouped the 46 representative isolates into five genotypic clusters, of which four of the clusters consisted of genotypically related isolates recovered from feed and fecal samples. The occurrence of genotypically related and, in some cases, clonal strains, including multidrug-resistant isolates in commercially processed feed and fecal samples, suggests the high significance of commercial feed as a potential vehicle ofSalmonellatransmission.
Xyn10A, a Thermostable Endoxylanase from
Acidothermus cellulolyticus
11B
Authors: Barabote Ravi D. , Parales Juanito V. , Guo Ying-Yi , Labavitch John M. , Parales Rebecca E. , Berry Alison M.
Published: September 2010

We cloned and purified the major family 10 xylanase (Xyn10A) from
Acidothermus cellulolyticus
11B. Xyn10A was active on oat spelt and birchwood xylans between 60°C and 100°C and between pH 4 and pH 8. The optimal activity was at 90°C and pH 6; specific activity and

K
m

for oat spelt xylan were 350 μmol xylose produced min
−1
mg of protein
−1
and 0.53 mg ml
−1
, respectively. Based on xylan cleavage patterns, Xyn10A is an endoxylanase, and its half-life at 90°C was approximately 1.5 h in the presence of xylan.
Evolutionary Relationships among Primary Endosymbionts of the Mealybug Subfamily Phenacoccinae (Hemiptera: Coccoidea: Pseudococcidae)
Authors: Gruwell Matthew E. , Hardy Nate B. , Gullan Penny J. , Dittmar Katharina
Published: September 2010

Mealybugs (Coccoidea: Pseudococcidae) are sap-sucking plant parasites that harbor bacterial endosymbionts within specialized organs. Previous studies have identified two subfamilies, Pseudococcinae and Phenacoccinae, within mealybugs and determined the primary endosymbionts (P-endosymbionts) of the Pseudococcinae to be
Betaproteobacteria
(“
Candidatus
Tremblaya princeps”) containing
Gammaproteobacteria
secondary symbionts. Here, the P-endosymbionts of phenacoccine mealybugs are characterized based on 16S rRNA from the bacteria of 20 species of phenacoccine mealybugs and four outgroup
Puto
species (Coccoidea: Putoidae) and aligned to more than 100 published 16S rRNA sequences from symbiotic and free-living bacteria. Phylogenetic analyses recovered three separate lineages of bacteria from the Phenacoccinae, and these are considered to be the P-endosymbionts of their respective mealybug hosts, with those from (i) the mealybug genus
Rastrococcus
belonging to the
Bacteroidetes
, (ii) the subterranean mealybugs, tribe Rhizoecini, also within
Bacteroidetes
, in a clade sister to cockroach endosymbionts (
Blattabacterium
), and (iii) the remaining Phenacoccinae within the
Betaproteobacteria
, forming a well-supported sister group to “
Candidatus
Tremblaya princeps.” Names are proposed for two strongly supported lineages: “
Candidatus
Brownia rhizoecola” for P-endosymbionts of Rhizoecini and “
Candidatus
Tremblaya phenacola” for P-endosymbionts of Phenacoccinae excluding
Rastrococcus
and Rhizoecini. Rates of nucleotide substitution among lineages of
Tremblaya
were inferred to be significantly faster than those of free-living
Betaproteobacteria
. Analyses also recovered a clade of
Gammaproteobacteria
, sister to the P-endosymbiont lineage of aphids (“
Candidatus
Buchnera aphidicola”), containing the endosymbionts of Putoidae, the secondary endosymbionts of pseudococcine mealybugs, and the endosymbionts of several other insect groups.
lacZ
Reporter System for Use in
Borrelia burgdorferi
Authors: Hayes Beth M. , Jewett Mollie W. , Rosa Patricia A.
Published: September 2010

Regulation of gene expression is critical for the ability of
Borrelia burgdorferi
to adapt to different environments during its natural infectious cycle. Reporter genes have been used successfully to study gene regulation in multiple organisms. We have introduced a
lacZ
gene into
B. burgdorferi
, and we show that
B. burgdorferi
produces a protein with detectable β-galactosidase activity in both liquid and solid media when
lacZ
is expressed from a constitutive promoter. Furthermore, when
lacZ
is expressed from the
ospC
promoter, β-galactosidase activity is detected only in
B. burgdorferi
clones that express
ospC
, and it accurately monitors endogenous gene expression. The addition of
lacZ
to the repertoire of genetic tools available for use in
B. burgdorferi
should contribute to a better understanding of how
B. burgdorferi
gene expression is regulated during the infectious cycle.
Isolation and Characterization of the Gene Cluster for Biosynthesis of the Thiopeptide Antibiotic TP-1161
Authors: Engelhardt Kerstin , Degnes Kristin F. , Zotchev Sergey B.
Published: September 2010

Recently, we isolated a new thiopeptide antibiotic, TP-1161, from the fermentation broth of a marine actinomycete typed as a member of the genus
Nocardiopsis
. Here we report the identification, isolation, and analysis of the TP-1161 biosynthetic gene cluster from this species. The gene cluster was identified by mining a draft genome sequence using the predicted structural peptide sequence of TP-1161. Functional assignment of a ∼16-kb genomic region revealed 13 open reading frames proposed to constitute the TP-1161 biosynthetic locus. While the typical core set of thiopeptide modification enzymes contains one cyclodehydratase/dehydrogenase pair, paralogous genes predicted to encode additional cyclodehydratases and dehydrogenases were identified. Although attempts at heterologous expression of the TP-1161 gene cluster in
Streptomyces coelicolor
failed, its identity was confirmed through the targeted gene inactivation in the original host.
Genetic Transformation and Mutagenesis via Single-Stranded DNA in the Unicellular, Diazotrophic Cyanobacteria of the Genus
Cyanothece
Authors: Min Hongtao , Sherman Louis A.
Published: September 2010

We describe a genetic system for producing specific gene knockouts in
Cyanothece
sp. strain PCC 7822 using a single-stranded DNA technique (B. Zorin, P. Hegemann, and I. Sizova, Eukaryot. Cell 4:1264-1272, 2005). The first fully segregated mutant was a Δ
nifK
mutant, and it was unable to grow on medium lacking combined nitrogen and produced virtually no hydrogen.
Covariation of Soil Bacterial Composition with Plant Rarity
Authors: Huguet Valérie , Rudgers Jennifer A.
Published: September 2010

Rare and common plants are predicted to host different rhizospheric microbes. To evaluate this prediction, we used terminal restriction fragment length polymorphism (T-RFLP) analysis to compare rhizospheric bacteria from eight native grass species whose local abundances in their natural ecosystem spanned a 15-fold range. We observed that locally rare versus common plants are associated with divergent bacterial communities.
Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates
Authors: Kostallas George , Samuelson Patrik
Published: September 2010

We have developed a sensitive and highly efficient whole-cell methodology for quantitative analysis and screening of protease activityin vivo. The method is based on the ability of a genetically encoded protease to rescue a coexpressed short-lived fluorescent substrate reporter from cytoplasmic degradation and thereby confer increased whole-cell fluorescence in proportion to the protease's apparent activity in theEscherichia colicytoplasm. We demonstrated that this system can reveal differences in the efficiency with which tobacco etch virus (TEV) protease processes different substrate peptides. In addition, when analyzingE. colicells expressing TEV protease variants that differed in terms of theirin vivosolubility, cells containing the most-soluble protease variant exhibited the highest fluorescence intensity. Furthermore, flow cytometry screening allowed for enrichment and subsequent identification of an optimal substrate peptide and protease variant from a large excess of cells expressing suboptimal variants (1:100,000). Two rounds of cell sorting resulted in a 69,000-fold enrichment and a 22,000-fold enrichment of the superior substrate peptide and protease variant, respectively. Our approach presents a new promising path forward for high-throughput substrate profiling of proteases, engineering of novel protease variants with desired properties (e.g., altered substrate specificity and improved solubility and activity), and identification of protease inhibitors.
Moderate Temperature Fluctuations Rapidly Reduce the Viability of
Ralstonia solanacearum
Race 3, Biovar 2, in Infected Geranium, Tomato, and Potato Plants
Authors: Scherf Jacob M. , Milling Annett , Allen Caitilyn
Published: September 2010

Most
Ralstonia solanacearum
strains are tropical plant pathogens, but race 3, biovar 2 (R3bv2), strains can cause bacterial wilt in temperate zones or tropical highlands where other strains cannot. R3bv2 is a quarantine pathogen in North America and Europe because of its potential to damage the potato industry in cooler climates. However, R3bv2 will not become established if it cannot survive temperate winters. Previous experiments showed that in water at 4°C, R3bv2 does not survive as long as native U.S. strains, but R3bv2 remains viable longer than U.S. strains in potato tubers at 4°C. To further investigate the effects of temperature on this high-concern pathogen, we assessed the ability of R3bv2 and a native U.S. strain to survive typical temperate winter temperature cycles of 2 days at 5°C followed by 2 days at −10°C. We measured pathogen survival in infected tomato and geranium plants, in infected potato tubers, and in sterile water. The population sizes of both strains declined rapidly under these conditions in all three plant hosts and in sterile water, and no culturable
R. solanacearum
cells were detected after five to seven temperature cycles in plant tissue. The fluctuations played a critical role in loss of bacterial viability, since at a constant temperature of −20°C, both strains could survive in infected geranium tissue for at least 6 months. These results suggest that even when sheltered in infected plant tissue, R3bv2 is unlikely to survive the temperature fluctuations typical of a northern temperate winter.
Novel Activity of
Rhodobacter sphaeroides
Spheroidene Monooxygenase CrtA Expressed in
Escherichia coli
Authors: Lee Pyung Cheon , Holtzapple Erik , Schmidt-Dannert Claudia
Published: September 2010

The spheroidene monooxygenase CrtA of
Rhodobacter sphaeroides
introduces a keto group and/or hydroxy group at the ends of nonnative substrates in
Escherichia coli
, resulting in the production of novel oxocarotenoids. The heme-containing CrtA is not a P450 enzyme but a new type of oxygenase.
New Insights into the Fructosyltransferase Activity ofSchwanniomyces occidentalisβ-Fructofuranosidase, Emerging from Nonconventional Codon Usage and Directed Mutation
Authors: Álvaro-Benito Miguel , de Abreu Miguel , Portillo Francisco , Sanz-Aparicio Julia , Fernández-Lobato María
Published: September 2010

Schwanniomyces occidentalisβ-fructofuranosidase (Ffase) releases β-fructose from the nonreducing ends of β-fructans and synthesizes 6-kestose and 1-kestose, both considered prebiotic fructooligosaccharides. Analyzing the amino acid sequence of this protein revealed that it includes a serine instead of a leucine at position 196, caused by a nonuniversal decoding of the unique mRNA leucine codon CUG. Substitution of leucine for Ser196 dramatically lowers the apparent catalytic efficiency (kcat/Km) of the enzyme (approximately 1,000-fold), but surprisingly, its transferase activity is enhanced by almost 3-fold, as is the enzymes' specificity for 6-kestose synthesis. The influence of 6 Ffase residues on enzyme activity was analyzed on both the Leu196/Ser196 backgrounds (Trp47, Asn49, Asn52, Ser111, Lys181, and Pro232). Only N52S and P232V mutations improved the transferase activity of the wild-type enzyme (about 1.6-fold). Modeling the transfructosylation products into the active site, in combination with an analysis of the kinetics and transfructosylation reactions, defined a new region responsible for the transferase specificity of the enzyme.
Molecular Evidence for Multiple Infections as Revealed by Typing of
Asaia
Bacterial Symbionts of Four Mosquito Species
Authors: Chouaia Bessem , Rossi Paolo , Montagna Matteo , Ricci Irene , Crotti Elena , Damiani Claudia , Epis Sara , Faye Ingrid , Sagnon N'Fale , Alma Alberto , Favia Guido , Daffonchio Daniele , Bandi Claudio
Published: September 2010

The recent increased detection of acetic acid bacteria (AAB) of the genus
Asaia
as symbionts of mosquitoes, such as
Anopheles
spp. and
Aedes
spp., prompted us to investigate the diversity of these symbionts and their relationships in different mosquito species and populations. Following cultivation-dependent and -independent techniques, we investigated the microbiota associated with four mosquito species,
Anopheles stephensi
,
Anopheles gambiae
,
Aedes aegypti
, and
Aedes albopictus
, which are important vectors of human and/or animal pathogens. Denaturing gradient gel electrophoresis (DGGE) analysis based on the 16S rRNA gene revealed the presence of several bacterial taxa, among which
Asaia
sequences were among the dominant in most of the samples. A collection of 281
Asaia
isolates in cell-free media was established from individuals belonging to the four species. The isolates were typed by internal transcribed spacer (ITS)-PCR, tRNA-PCR, BOX-PCR, and randomly amplified polymorphic DNA (RAPD)-PCR, revealing that different
Asaia
strains are present in different mosquito populations, and even in single individuals.
EseG, an Effector of the Type III Secretion System of
Edwardsiella tarda
, Triggers Microtubule Destabilization
Authors: Xie Hai Xia , Yu Hong Bing , Zheng Jun , Nie Pin , Foster Leonard J. , Mok Yu-Keung , Finlay B. Brett , Leung Ka Yin
Published: September 2010

Edwardsiella tarda
is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and both gastrointestinal and extraintestinal infections in humans. A type III secretion system (T3SS) was recently shown to contribute to pathogenesis, since deletions of various T3SS genes increased the 50% lethal dose (LD
50
) by about 1 log unit in the blue gourami infection model. In this study, we report EseG as the first identified effector protein of T3SS. EseG shares partial homology with two
Salmonella
T3SS effectors (SseG and SseF) over a conserved domain (amino acid residues 142 to 192). The secretion of EseG is dependent on a functional T3SS and, in particular, requires the chaperone EscB. Experiments using TEM-1 β-lactamase as a fluorescence-based reporter showed that EseG was translocated into HeLa cells at 35°C. Fractionation of infected HeLa cells demonstrated that EseG was localized to the host membrane fraction after translocation. EseG is able to disassemble microtubule structures when overexpressed in mammalian cells. This phenotype may require a conserved motif of EseG (EseG
142-192
), since truncated versions of EseG devoid of this motif lose their ability to cause microtubule destabilization. By demonstrating the function of EseG, our study contributes to the understanding of
E. tarda
pathogenesis. Moreover, the approach established in this study to identify type III effectors can be used to identify and characterize more type III and possible type VI effectors in
Edwardsiella
.
The Type VI Secretion System Plays a Role in Type 1 Fimbria Expression and Pathogenesis of an Avian Pathogenic
Escherichia coli
Strain
Authors: de Pace Fernanda , Nakazato Gerson , Pacheco Alline , Boldrin de Paiva Jacqueline , Sperandio Vanessa , Dias da Silveira Wanderley
Published: September 2010

Avian pathogenic
Escherichia coli
(APEC) strains frequently cause extraintestinal infections and are responsible for significant economic losses in the poultry industry worldwide. APEC isolates are closely related to human extraintestinal pathogenic
E. coli
(ExPEC) strains and may also act as pathogens for humans. Known APEC virulence factors include adhesins such as type 1 fimbriae and curli, iron acquisition systems, and cytotoxins. Here we show that APEC strain SEPT362, isolated from a septicemic hen, expresses a type VI secretion system (T6SS); causes cytoskeleton rearrangements; and invades epithelial cells, replicates within macrophages, and causes lethal disease in chicks. To assess the contribution of the T6SS to SEPT362 pathogenesis, we generated two mutants,
hcp
(which encodes a protein suggested to be both secreted and a structural component of the T6SS) and
clpV
(encoding the T6SS ATPase). Both mutants showed decreased adherence and actin rearrangement on epithelial cells. However, only the
hcp
mutant presented a mild decrease in its ability to invade epithelial cells, and none of these mutants were defective for intramacrophage replication. Transcriptome studies showed that the level of expression of type 1 fimbriae was decreased in these mutants, which may account for the diminished adhesion and invasion of epithelial cells. The T6SS seems to be important for the disease process, given that both mutants were attenuated for infection in chicks. These results suggest that the T6SS influences the expression of type 1 fimbriae and contributes to APEC pathogenesis.
Znu Is the Predominant Zinc Importer in
Yersinia pestis
during
In Vitro
Growth but Is Not Essential for Virulence
Authors: Desrosiers Daniel C. , Bearden Scott W. , Mier Ildefonso , Abney Jennifer , Paulley James T. , Fetherston Jacqueline D. , Salazar Juan C. , Radolf Justin D. , Perry Robert D.
Published: September 2010

Little is known about Zn homeostasis in
Yersinia pestis
, the plague bacillus. The Znu ABC transporter is essential for zinc (Zn) uptake and virulence in a number of bacterial pathogens. Bioinformatics analysis identified ZnuABC as the only apparent high-affinity Zn uptake system in
Y. pestis
. Mutation of
znuACB
caused a growth defect in Chelex-100-treated PMH2 growth medium, which was alleviated by supplementation with submicromolar concentrations of Zn. Use of transcriptional reporters confirmed that Zur mediated Zn-dependent repression and that it can repress gene expression in response to Zn even in the absence of Znu. Virulence testing in mouse models of bubonic and pneumonic plague found only a modest increase in survival in low-dose infections by the
znuACB
mutant. Previous studies of cluster 9 (C9) transporters suggested that Yfe, a well-characterized C9 importer for manganese (Mn) and iron in
Y. pestis
, might function as a second, high-affinity Zn uptake system. Isothermal titration calorimetry revealed that YfeA, the solute-binding protein component of Yfe, binds Mn and Zn with comparably high affinities (dissociation constants of 17.8 ± 4.4 nM and 6.6 ± 1.2 nM, respectively), although the complete Yfe transporter could not compensate for the loss of Znu in
in vitro
growth studies. Unexpectedly, overexpression of Yfe interfered with the
znu
mutant's ability to grow in low concentrations of Zn, while excess Zn interfered with the ability of Yfe to import iron at low concentrations; these results suggest that YfeA can bind Zn in the bacterial cell but that Yfe is incompetent for transport of the metal. In addition to Yfe, we have now eliminated MntH, FetMP, Efe, Feo, a substrate-binding protein, and a putative nickel transporter as the unidentified, secondary Zn transporter in
Y. pestis.
Unlike other bacterial pathogens,
Y. pestis
does not require Znu for high-level infectivity and virulence; instead, it appears to possess a novel class of transporter, which can satisfy the bacterium's Zn requirements under
in vivo
metal-limiting conditions. Our studies also underscore the need for bacterial cells to balance binding and transporter specificities within the periplasm in order to maintain transition metal homeostasis.
Serotype-Related Variation in Susceptibility to Complement Deposition and Opsonophagocytosis among Clinical Isolates of
Streptococcus pneumoniae
Authors: Melin Merit , Trzciński Krzysztof , Antonio Martin , Meri Seppo , Adegbola Richard , Kaijalainen Tarja , Käyhty Helena , Väkeväinen Merja
Published: September 2010

The polysaccharide capsule is a major virulence factor of
Streptococcus pneumoniae
; it affects complement resistance and shields the bacterium from phagocytes. Certain capsular serotypes appear to be better able to cause invasive disease than others. Serotypes 1 and 5 are common causes of invasive disease but are rarely isolated from healthy carriers, whereas serotypes 6B and 23F are more frequently isolated from carriage than invasive disease. We have recently shown that serotypes 6B and 19F differ in resistance to complement C3 deposition and opsonophagocytic killing. In this study we assessed the complement resistance and susceptibility to opsonophagocytosis of several other serotypes targeted by the pneumococcal conjugate vaccines. Clinical isolates of serotypes 1, 4, 5, 14, 18C, and 23F were tested along reference strains of corresponding capsular types. The concentration of anticapsular antibodies required for opsonophagocytic killing correlated inversely with C3 deposition on the serotype. Serotype 1 was the most resistant of the clinical isolates to C3 deposition and, along with serotypes 5 and 19F, required the highest concentration of capsule antibodies for opsonophagocytic killing, whereas serotype 23F was the most sensitive to opsonophagocytosis. Sensitivity to C3 deposition and opsonophagocytosis was associated with serotype-specific mortality of invasive pneumococcal disease, suggesting that the primary pathogens, such as serotypes 1 and 5, are more resistant to complement and require a higher concentration of capsule antibodies for opsonophagocytic killing than the opportunistic serotypes such as 6B and 23F, which are associated with a more severe disease outcome.