bluuebunny/crossref_metadata_2025_split huggingface dataset:
Degradation of Methanethiol by Methylotrophic Methanogenic Archaea in a Lab-Scale Upflow Anaerobic Sludge Blanket Reactor
Authors: de Bok F. A. M. , van Leerdam R. C. , Lomans B. P. , Smidt H. , Lens P. N. L. , Janssen A. J. H. , Stams A. J. M. 
Published: December 2006

ABSTRACT

In a lab-scale upflow anaerobic sludge blanket reactor inoculated with granular sludge from a full-scale wastewater treatment plant treating paper mill wastewater, methanethiol (MT) was degraded at 30°C to H
2
S, CO
2
, and CH
4
. At a hydraulic retention time of 9 h, a maximum influent concentration of 6 mM MT was applied, corresponding to a volumetric loading rate of 16.5 mmol liter
−1
day
−1
. The archaeal community within the reactor was characterized by anaerobic culturing and denaturing gradient gel electrophoresis analysis, cloning, and sequencing of 16S rRNA genes and quantitative PCR. Initially, MT-fermenting methanogenic archaea related to members of the genus
Methanolobus
were enriched in the reactor. Later, they were outcompeted by
Methanomethylovorans hollandica
, which was detected in aggregates but not inside the granules that originated from the inoculum, the microbial composition of which remained fairly unchanged. Possibly other species within the
Methanosarcinacaea
also contributed to the fermentation of MT, but they were not enriched by serial dilution in liquid media. The archaeal community within the granules, which was dominated by
Methanobacterium beijingense
, did not change substantially during the reactor operation. Some of the species related to
Methanomethylovorans hollandica
were enriched by serial dilutions, but their growth rates were very low. Interestingly, the enrichments could be sustained only in the presence of MT and did not utilize any of the other typical substrates for methylotrophic methanogens, such as methanol, methyl amine, or dimethylsulfide.
Escherichia coli
Biofilms Formed under Low-Shear Modeled Microgravity in a Ground-Based System
Authors: Lynch S. V. , Mukundakrishnan K. , Benoit M. R. , Ayyaswamy P. S. , Matin A. 
Published: December 2006

ABSTRACT

Bacterial biofilms cause chronic diseases that are difficult to control. Since biofilm formation in space is well documented and planktonic cells become more resistant and virulent under modeled microgravity, it is important to determine the effect of this gravity condition on biofilms. Inclusion of glass microcarrier beads of appropriate dimensions and density with medium and inoculum, in vessels specially designed to permit ground-based investigations into aspects of low-shear modeled microgravity (LSMMG), facilitated these studies. Mathematical modeling of microcarrier behavior based on experimental conditions demonstrated that they satisfied the criteria for LSMMG conditions. Experimental observations confirmed that the microcarrier trajectory in the LSMMG vessel concurred with the predicted model. At 24 h, the LSMMG
Escherichia coli
biofilms were thicker than their normal-gravity counterparts and exhibited increased resistance to the general stressors salt and ethanol and to two antibiotics (penicillin and chloramphenicol). Biofilms of a mutant of
E. coli
, deficient in σ
s
, were impaired in developing LSMMG-conferred resistance to the general stressors but not to the antibiotics, indicating two separate pathways of LSMMG-conferred resistance.
Bioinformatic, Genetic, and Biochemical Evidence that Some Glycoside Hydrolase Family 42 β-Galactosidases Are Arabinogalactan Type I Oligomer Hydrolases
Authors: Shipkowski Stephanie , Brenchley Jean E. 
Published: December 2006

ABSTRACT

Glycoside hydrolases are organized into glycoside hydrolase families (GHFs) and within this larger group, the β-galactosidases are members of four families: 1, 2, 35, and 42. Most genes encoding GHF 42 enzymes are from prokaryotes unlikely to encounter lactose, suggesting a different substrate for these enzymes. In search of this substrate, we analyzed genes neighboring GHF 42 genes in databases and detected an arrangement implying that these enzymes might hydrolyze oligosaccharides released by GHF 53 enzymes from arabinogalactan type I, a pectic plant polysaccharide. Because
Bacillus subtilis
has adjacent GHF 42 and GHF 53 genes, we used it to test the hypothesis that a GHF 42 enzyme (LacA) could act on the oligosaccharides released by a GHF 53 enzyme (GalA) from galactan. We cloned these genes, plus a second GHF 42 gene from
B. subtilis
,
yesZ
, into
Escherichia coli
and demonstrated that cells expressing LacA with GalA gained the ability to use galactan as a carbon source. We constructed
B. subtilis
mutants and showed that the increased β-galactosidase activity generated in response to the addition of galactan was eliminated by inactivating
lacA
or
galA
but unaffected by the inactivation of
yesZ
. As further demonstration, we overexpressed the LacA and GalA proteins in
E. coli
and demonstrated that these enzymes degrade galactan in vitro as assayed by thin-layer chromatography. Our work provides the first in vivo evidence for a function of some GHF 42 β-galactosidases. Similar functions for other β-galactosidases in both GHFs 2 and 42 are suggested by genomic data.
Transformation of the Green Alga
Haematococcus pluvialis
with a Phytoene Desaturase for Accelerated Astaxanthin Biosynthesis
Authors: Steinbrenner Jens , Sandmann Gerhard 
Published: December 2006

ABSTRACT

Astaxanthin is a high-value carotenoid which is used as a pigmentation source in fish aquaculture. Additionally, a beneficial role of astaxanthin as a food supplement for humans has been suggested. The unicellular alga
Haematococcus pluvialis
is a suitable biological source for astaxanthin production. In the context of the strong biotechnological relevance of
H. pluvialis
, we developed a genetic transformation protocol for metabolic engineering of this green alga. First, the gene coding for the carotenoid biosynthesis enzyme phytoene desaturase was isolated from
H. pluvialis
and modified by site-directed mutagenesis, changing the leucine codon at position 504 to an arginine codon. In an in vitro assay, the modified phytoene desaturase was still active in conversion of phytoene to ζ-carotene and exhibited 43-fold-higher resistance to the bleaching herbicide norflurazon. Upon biolistic transformation using the modified phytoene desaturase gene as a reporter and selection with norflurazon, integration into the nuclear genome of
H. pluvialis
and phytoene desaturase gene and protein expression were demonstrated by Southern, Northern, and Western blotting, respectively, in 11 transformants. Some of the transformants had a higher carotenoid content in the green state, which correlated with increased nonphotochemical quenching. This measurement of chlorophyll fluorescence can be used as a screening procedure for stable transformants. Stress induction of astaxanthin biosynthesis by high light showed that there was accelerated accumulation of astaxanthin in one of the transformants compared to the accumulation in the wild type. Our results strongly indicate that the modified phytoene desaturase gene is a useful tool for genetic engineering of carotenoid biosynthesis in
H. pluvialis
.
Identification of Pyruvate Carboxylase Genes in
Pseudomonas aeruginosa
PAO1 and Development of a
P. aeruginosa
-Based Overexpression System for α
4
- and α
4
β
4
-Type Pyruvate Carboxylases
Authors: Lai Huafang , Kraszewski Jessica L. , Purwantini Endang , Mukhopadhyay Biswarup 
Published: December 2006

ABSTRACT

Pyruvate carboxylase (PYC) is an ecologically, medically, and industrially important enzyme. It is widespread in all three domains of life, the archaea, bacteria, and eukarya. PYC catalyzes ATP-dependent carboxylation of pyruvate to oxaloacetate. Detailed structure-function studies of this enzyme have been hampered due to the unavailability of a facile recombinant overexpression system. Except for the α
4
enzyme from a thermophilic
Bacillus
species,
Escherichia coli
has been unsuitable for overexpression of PYCs. We show that a
Pseudomonas aeruginosa
strain carrying the T7 polymerase gene can serve as a host for the overexpression of
Mycobacterium smegmatis
α
4
PYC and
Pseudomonas aeruginosa
α
4
β
4
PYC under the control of the T7 promoter from a broad-host-range conjugative plasmid. Overexpression occurred both in aerobic (LB medium) and nitrate-respiring anaerobic (LB medium plus glucose and nitrate) cultures. The latter system presented a simpler option because it involved room temperature cultures in stationary screw-cap bottles. We also developed a
P. aeruginosa Δpyc
strain that allowed the expression of recombinant PYCs in the absence of the native enzyme. Since
P. aeruginosa
can be transformed genetically and lysed for cell extract preparation rather easily, our system will facilitate site-directed mutagenesis, kinetics, X-ray crystallographic, and nuclear magnetic resonance-based structure-function analysis of PYCs. During this work we also determined that, contrary to a previous report (C. K. Stover et al., Nature 406:959-964, 2000), the open reading frame (ORF) PA1400 does not encode a PYC in
P. aeruginosa
. The α
4
β
4
PYC of this organism was encoded by the ORFs PA5436 and PA5435.
Longitudinal Analysis of Tick Densities and
Borrelia
,
Anaplasma
, and
Ehrlichia
Infections of
Ixodes ricinus
Ticks in Different Habitat Areas in The Netherlands
Authors: Wielinga Peter R. , Gaasenbeek Cor , Fonville Manoj , de Boer Albert , de Vries Ankje , Dimmers Wim , Akkerhuis Op Jagers Gerard , Schouls Leo M. , Borgsteede Fred , van der Giessen Joke W. B. 
Published: December 2006

ABSTRACT

From 2000 to 2004, ticks were collected by dragging a blanket in four habitat areas in The Netherlands: dunes, heather, forest, and a city park. Tick densities were calculated, and infection with
Borrelia burgdorferi
and
Anaplasma
and
Ehrlichia
species was investigated by reverse line blot analysis. The lowest tick density was observed in the heather area (1 to 8/100 m
2
). In the oak forest and city park, the tick densities ranged from 26 to 45/100 m
2
. The highest tick density was found in the dune area (139 to 551/100 m
2
). The infection rates varied significantly for the four study areas and years, ranging from 0.8 to 11. 5% for
Borrelia
spp. and 1 to 16% for
Ehrlichia
or
Anaplasma
(
Ehrlichia/Anaplasma
) spp.
Borrelia
infection rates were highest in the dunes, followed by the forest, the city park, and heather area. In contrast,
Ehrlichia/Anaplasma
was found most often in the forest and less often in the city park. The following
Borrelia
species were found:
Borrelia
sensu lato strains not identified to the species level (2.5%),
B. afzelii
(2.5%),
B. valaisiana
(0.9%),
B. burgdorferi
sensu stricto (0.13%), and
B. garinii
(0.13%). For
Ehrlichia/Anaplasma
species,
Ehrlichia
and
Anaplasma
spp. not identified to the species level (2.5%),
Anaplasma schotti
variant (3.5%),
Anaplasma phagocytophilum
variant (0.3%), and
Ehrlichia canis
(0.19%) were found.
E. canis
is reported for the first time in ticks in The Netherlands in this study.
Borrelia lusitaniae
,
Ehrlichia chaffeensis
, and the human granylocytic anaplasmosis agent were not detected. About 1.6% of the ticks were infected with both
Borrelia
and
Ehrlichia/Anaplasma
, which was higher than the frequency predicted from the individual infection rates, suggesting hosts with multiple infections or a possible selective advantage of coinfection.
A Real-Time PCR Method for Quantifying Viable
Ascaris
Eggs Using the First Internally Transcribed Spacer Region of Ribosomal DNA
Authors: Pecson Brian M. , Barrios José Antonio , Johnson David R. , Nelson Kara L. 
Published: December 2006

ABSTRACT

Worldwide, 1.4 billion people are infected with the intestinal worm
Ascaris lumbricoides
. As a result,
Ascaris
eggs are commonly found in wastewater and sludges. The current microscopy method for detecting viable
Ascaris
eggs is time- and labor-intensive. The goal of this study was to develop a real-time quantitative PCR (qPCR) method to determine the levels of total and viable
Ascaris
eggs in laboratory solutions using the first internally transcribed spacer (ITS-1) region of ribosomal DNA (rDNA) and rRNA. ITS-1 rDNA levels were proportional to
Ascaris
egg cell numbers, increasing as eggs developed from single cells to mature larvae and ultimately reaching a constant level per egg. Treatments causing >99% inactivation (high heat, moderate heat, ammonia, and UV) eliminated this increase in ITS-1 rDNA levels and caused decreases that were dependent on the treatment type. By taking advantage of this difference in ITS-1 rDNA level between viable, larvated eggs and inactivated, single-celled eggs, qPCR results were used to develop inactivation profiles for the different treatments. No statistical difference from the standard microscopy method was found in 75% of the samples (12 of 16). ITS-1 rRNA was detected only in samples containing viable eggs, but the levels were more variable than rDNA levels and ITS-1 rRNA could not be used for quantification. The detection limit of the rDNA-based method was approximately one larvated egg or 90 single-celled eggs; the detection limit for the rRNA-based method was several orders of magnitude higher. The rDNA qPCR method is promising for both research and regulatory applications.
Hypervirulence of a Rough Variant of the
Mycobacterium abscessus
Type Strain
Authors: Catherinot E. , Clarissou J. , Etienne G. , Ripoll F. , Emile J.-F. , Daffé M. , Perronne C. , Soudais C. , Gaillard J.-L. , Rottman M. 
Published: December 2006

ABSTRACT

We isolated a rough variant of
Mycobacterium abscessus
CIP 104536T during experimental infection of mice. We show that this variant has lost the ability to produce glycopeptidolipids, is hyperlethal for C57BL/6 mice infected intravenously, and induces a strong tumor necrosis factor-alpha response by murine monocyte-derived macrophages.
Protective Effect of the Nramp1 BB Genotype againstBrucella abortusin the Water Buffalo (Bubalus bubalis)
Authors: Capparelli Rosanna , Alfano Flora , Amoroso Maria Grazia , Borriello Giorgia , Fenizia Domenico , Bianco Antonio , Roperto Sante , Roperto Franco , Iannelli Domenico 
Published: December 2006

ABSTRACTWe tested 413 water buffalo cows (142 cases and 271 controls) for the presence of anti-Brucella abortusantibodies (by the skin test, the agglutination test, and the complement fixation test) and theNramp1genotype (by capillary electrophoresis). Four alleles (Nramp1A, -B, -C, and -D) were detected in the 3′ untranslated region of theNramp1gene. TheBBgenotype was represented among only controls, providing evidence that this genotype confers resistance toBrucella abortus. The monocytes from the BB (resistant) subjects displayed a higher basal level ofNramp1mRNA and a lower number of viable intracellular bacteria than did the monocytes from AA (susceptible) subjects. The higher basal level of the antibacterial protein Nramp1 most probably provides the BB animals with the possibility of controlling bacteria immediately after their entry inside the cell.
Differential Abilities of Capsulated and Noncapsulated
Staphylococcus aureus
Isolates from Diverse
agr
Groups To Invade Mammary Epithelial Cells
Authors: Buzzola Fernanda R. , Alvarez Lucía P. , Tuchscherr Lorena P. N. , Barbagelata María S. , Lattar Santiago M. , Calvinho Luis , Sordelli Daniel O. 
Published: December 2006

ABSTRACT

Staphylococcus aureus
is the bacterium most frequently isolated from milk of bovines with mastitis. Four allelic groups, which interfere with the regulatory activities among the different groups, have been identified in the accessory gene regulator (
agr
) system. The aim of this study was to ascertain the prevalence of the different
agr
groups in capsulated and noncapsulated
S. aureus
bacteria isolated from mastitic bovines in Argentina and whether a given
agr
group was associated with MAC-T cell invasion and in vivo persistence. Eighty-eight percent of the bovine
S. aureus
strains were classified in
agr
group I. The remainder belonged in
agr
groups II, III, and IV (2, 8, and 2%, respectively). By restriction fragment length polymorphism analysis after PCR amplification of the
agr
locus variable region, six
agr
restriction types were identified. All
agr
group I strains presented a unique allele (A/1), whereas strains from groups II, III, and IV exhibited more diversity. Bovine
S. aureus
strains defined as being in
agr
group I (capsulated or noncapsulated) showed significantly increased abilities to be internalized within MAC-T cells, compared with isolates from
agr
groups II, III, and IV.
agr
group II or IV
S. aureus
strains were cleared more efficiently than
agr
group I strains from the murine mammary gland. The results suggest that
agr
group I
S. aureus
strains are more efficiently internalized within epithelial cells and can persist in higher numbers in mammary gland tissue than
S. aureus
strains classified in
agr
group II, III, or IV.
The Early Transcriptional Response of Human Granulocytes to Infection withCandida albicansIs Not Essential for Killing but Reflects Cellular Communications
Authors: Fradin Chantal , Mavor Abigail L. , Weindl Günther , Schaller Martin , Hanke Karin , Kaufmann Stefan H. E. , Mollenkopf Hans , Hube Bernhard 
Published: December 2006

ABSTRACTCandida albicansis a polymorphic opportunistic fungus that can cause life-threatening systemic infections following hematogenous dissemination in patients susceptible to nosocomial infection. Neutrophils form part of the innate immune response, which is the first line of defense against microbes and is particularly important inC. albicansinfections. To compare the transcriptional response of leukocytes exposed toC. albicans, we investigated the expression of key cytokine genes in polymorphonuclear and mononuclear leukocytes after incubation withC. albicansfor 1 h. Isolated mononuclear cells expressed high levels of genes encoding proinflammatory signaling molecules, whereas neutrophils exhibited much lower levels, similar to those observed in whole blood. The global transcriptional profile of neutrophils was examined by using an immunology-biased human microarray to determine whether different morphological forms or the viability ofC. albicansaltered the transcriptome. Hyphal cells appeared to have the broadest effect, although the most strongly induced genes were regulated independently of morphology or viability. These genes were involved in proinflammatory cell-cell signaling, cell signal transduction, and cell growth. Generally, genes encoding known components of neutrophil granules showed no upregulation at this time point; however, lactoferrin, a well-known candidacidal peptide, was secreted by neutrophils. Addition to inhibitors of RNA or protein de novo synthesis did not influence the killing activity within 30 min. These results support the general notion that neutrophils do not require gene transcription to mount an immediate and direct attack against microbes. However, neutrophils exposed toC. albicansexpress genes involved in communication with other immune cells.
Effect of Site-Specific Mutations in Different Phosphotransfer Domains of the Chemosensory Protein ChpA onPseudomonas aeruginosaMotility
Authors: Leech Andrew J. , Mattick John S. 
Published: December 2006

ABSTRACTThe virulence ofPseudomonas aeruginosaand other surface pathogens involves the coordinate expression of a wide range of virulence determinants, including type IV pili. These surface filaments are important for the colonization of host epithelial tissues and mediate bacterial attachment to, and translocation across, surfaces by a process known as twitching motility. This process is controlled in part by a complex signal transduction system whose central component, ChpA, possesses nine potential sites of phosphorylation, including six histidine-containing phosphotransfer (HPt) domains, one serine-containing phosphotransfer domain, one threonine-containing phosphotransfer domain, and one CheY-like receiver domain. Here, using site-directed mutagenesis, we show that normal twitching motility is entirely dependent on the CheY-like receiver domain and partially dependent on two of the HPt domains. Moreover, under different assay conditions, point mutations in several of the phosphotransfer domains of ChpA give rise to unusual “swarming” phenotypes, possibly reflecting more subtle perturbations in the control ofP. aeruginosamotility that are not evident from the conventional twitching stab assay. Together, these results suggest that ChpA plays a central role in the complex regulation of type IV pilus-mediated motility inP. aeruginosa.
Agrobacterium tumefaciens soxR
Is Involved in Superoxide Stress Protection and Also Directly Regulates Superoxide-Inducible Expression of Itself and a Target Gene
Authors: Eiamphungporn Warawan , Charoenlap Nisanart , Vattanaviboon Paiboon , Mongkolsuk Skorn 
Published: December 2006

ABSTRACT

Inactivation of
Agrobacterium tumefaciens soxR
increases sensitivity to superoxide generators.
soxR
expression is highly induced by superoxide stress and is autoregulated. SoxR also directly regulates the superoxide-inducible expression of atu5152. Taken together, the physiological role of
soxR
and the mechanism by which it regulates expression of target genes make the
A. tumefaciens
SoxR system different from other bacterial systems.
4-Chlorobenzoate Uptake in
Comamonas
sp. Strain DJ-12 Is Mediated by a Tripartite ATP-Independent Periplasmic Transporter
Authors: Chae Jong-Chan , Zylstra Gerben J. 
Published: December 2006

ABSTRACT

The
fcb
gene cluster involved in the hydrolytic dehalogenation of 4-chlorobenzoate is organized in the order
fcbB
-
fcbA
-
fcbT1
-
fcbT2
-
fcbT3
-
fcbC
in
Comamonas
sp. strain DJ-12. The genes are operonic and inducible with 4-chloro-, 4-iodo-, and 4-bromobenzoate. The
fcbT1
,
fcbT2
, and
fcbT3
genes encode a transporter in the secondary TRAP (tripartite ATP-independent periplasmic) family. An
fcbT1T2T3
knockout mutant shows a much slower growth rate on 4-chlorobenzoate compared to the wild type. 4-Chlorobenzoate is transported into the wild-type strain five times faster than into the
fcbT1T2T3
knockout mutant. Transport of 4-chlorobenzoate shows significant inhibition by 4-bromo-, 4-iodo-, and 4-fluorobenzoate and mild inhibition by 3-chlorobenzoate, 2-chlorobenzoate, 4-hydroxybenzoate, 3-hydroxybenzoate, and benzoate. Uptake of 4-chlorobenzoate is significantly inhibited by ionophores which collapse the proton motive force.
The UreEF Fusion Protein Provides a Soluble and Functional Form of the UreF Urease Accessory Protein
Authors: Kim Jong Kyong , Mulrooney Scott B. , Hausinger Robert P. 
Published: December 2006

ABSTRACT

Four accessory proteins (UreD, UreE, UreF, and UreG) are typically required to form the nickel-containing active site in the urease apoprotein (UreABC). Among the accessory proteins, UreD and UreF have been elusive targets for biochemical and structural characterization because they are not overproduced as soluble proteins. Using the best-studied urease system, in which the
Klebsiella aerogenes
genes are expressed in
Escherichia coli
, a translational fusion of
ureE
and
ureF
was generated. The UreEF fusion protein was overproduced as a soluble protein with a convenient tag involving the His-rich region of UreE. The fusion protein was able to form a UreD(EF)G-UreABC complex and to activate urease in vivo, and it interacted with UreD-UreABC in vitro to form a UreD(EF)-UreABC complex. While the UreF portion of UreEF is fully functional, the fusion significantly affected the role of the UreE portion by interrupting its dimerization and altering its metal binding properties compared to those of the wild-type UreE. Analysis of a series of UreEF deletion mutants revealed that the C terminus of UreF is required to form the UreD(EF)G-UreABC complex, while the N terminus of UreF is essential for activation of urease.