bluuebunny/crossref_metadata_2025 huggingface dataset:
Failure To Detect
Helicobacter pylori
DNA in Drinking and Environmental Water in Dhaka, Bangladesh, Using Highly Sensitive Real-Time PCR Assays
Authors: Janzon Anders , Sjöling Åsa , Lothigius Åsa , Ahmed Dilruba , Qadri Firdausi , Svennerholm Ann-Mari
Published: March 2009

The main transmission pathway of
Helicobacter pylori
has not been determined, but several reports have described detection of
H. pylori
DNA in drinking and environmental water, suggesting that
H. pylori
may be waterborne. To address this possibility, we developed, tested, and optimized two complementary
H. pylori
-specific real-time PCR assays for quantification of
H. pylori
DNA in water. The minimum detection level of the assays including collection procedures and DNA extraction was shown to be approximately 250
H. pylori
genomes per water sample. Using our assays, we then analyzed samples of drinking and environmental water (
n
= 75) and natural water biofilms (
n
= 21) from a high-endemicity area in Bangladesh. We could not identify
H. pylori
DNA in any of the samples, even though other pathogenic bacteria have been found previously in the same water samples by using the same methodology. A series of control experiments were performed to ensure that the negative results were not falsely caused by PCR inhibition, nonspecific assays, degradation of template DNA, or low detection sensitivity. Our results suggest that it is unlikely that the predominant transmission route of
H. pylori
in this area is waterborne.
Effect of Lake Trophic Status and Rooted Macrophytes on Community Composition and Abundance of Ammonia-Oxidizing Prokaryotes in Freshwater Sediments
Authors: Herrmann Martina , Saunders Aaron M. , Schramm Andreas
Published: March 2009

Communities of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in freshwater sediments and those in association with the root system of the macrophyte species
Littorella uniflora
,
Juncus bulbosus
, and
Myriophyllum alterniflorum
were compared for seven oligotrophic to mesotrophic softwater lakes and acidic heathland pools. Archaeal and bacterial ammonia monooxygenase alpha-subunit (
amoA
) gene diversity increased from oligotrophic to mesotrophic sites; the number of detected operational taxonomic units was positively correlated to ammonia availability and pH and negatively correlated to sediment C/N ratios. AOA communities could be grouped according to lake trophic status and pH; plant species-specific communities were not detected, and no grouping was apparent for AOB communities. Relative abundance, determined by quantitative PCR targeting
amoA
, was always low for AOB (<0.05% of all prokaryotes) and slightly higher for AOA in unvegetated sediment and AOA in association with
M. alterniflorum
(0.01 to 2%), while AOA accounted for up to 5% in the rhizospheres of
L. uniflora
and
J. bulbosus
. These results indicate that (i) AOA are at least as numerous as AOB in freshwater sediments, (ii) aquatic macrophytes with substantial release of oxygen and organic carbon into their rhizospheres, like
L. uniflora
and
J. bulbosus
, increase AOA abundance; and (iii) AOA community composition is generally determined by lake trophy, not by plant species-specific interactions.
Negative Correlation between Individual-Insect-Level Virulence and Colony-Level Virulence of
Paenibacillus larvae
, the Etiological Agent of American Foulbrood of Honeybees
Authors: Rauch Sandra , Ashiralieva Ainura , Hedtke Kati , Genersch Elke
Published: March 2009

Paenibacillus larvae
is the etiological agent of American foulbrood (AFB) in honeybees. Recently, different genotypes of
P. larvae
(ERIC I to ERIC IV) were defined, and it was shown that these genotypes differ inter alia in their virulence on the larval level. On the colony level, bees mitigate AFB through the hygienic behavior of nurse bees. Therefore, we investigated how the hygienic behavior shapes
P. larvae
virulence on the colony level. Our results indicate that
P. larvae
virulence on the larval level and that on the colony level are negatively correlated.
Influence of Stress on Single-Cell Lag Time and Growth Probability for
Listeria monocytogenes
in Half Fraser Broth
Authors: Dupont Claire , Augustin Jean-Christophe
Published: March 2009

The impacts of 12 common food industry stresses on the single-cell growth probability and single-cell lag time distribution of
Listeria monocytogenes
were determined in half Fraser broth, the primary enrichment broth of the International Organization for Standardization detection method. First, it was determined that the ability of a cell to multiply in half Fraser broth is conditioned by its history (the probability for a cell to multiply can be decreased to 0.05), meaning that, depending on the stress in question, the risk of false-negative samples can be very high. Second, it was established that when cells are injured, the single-cell lag times increase in mean and in variability and that this increase represents a true risk of not reaching the detection threshold of the method in the enrichment broth. No relationship was observed between the impact on single-cell lag times and that on growth probabilities. These results emphasize the importance of taking into account the physiological state of the cells when evaluating the performance of methods to detect pathogens in food.
Acetobacter tropicalisIs a Major Symbiont of the Olive Fruit Fly (Bactrocera oleae)
Authors: Kounatidis Ilias , Crotti Elena , Sapountzis Panagiotis , Sacchi Luciano , Rizzi Aurora , Chouaia Bessem , Bandi Claudio , Alma Alberto , Daffonchio Daniele , Mavragani-Tsipidou Penelope , Bourtzis Kostas
Published: March 2009

Following cultivation-dependent and -independent techniques, we investigated the microbiota associated withBactrocera oleae, one of the major agricultural pests in olive-producing countries. Bacterial 16S rRNA gene libraries and ultrastructural analyses revealed the presence of several bacterial taxa associated with this insect, among whichAcetobacter tropicaliswas predominant. The recent increased detection of acetic acid bacteria as symbionts of other insect model organisms, such asAnopheles stephensi(G. Favia et al., Proc. Natl. Acad. Sci. USA 104:9047-9051, 2007) orDrosophila melanogaster(C. R. Cox and M. S. Gilmore, Infect. Immun. 75:1565-1576, 2007), prompted us to investigate the association established betweenA. tropicalisandB. oleae. Using anA. tropicalis-specific PCR assay, the symbiont was detected in all insects tested originating from laboratory stocks or field-collected from different locations in Greece. This acetic acid bacterium was successfully established in cell-free medium, and typing analyses, carried out on a collection of isolates, revealed that differentA. tropicalisstrains are present in fly populations. The capability to colonize and lodge in the digestive system of both larvae and adults and in Malpighian tubules of adults was demonstrated by using a strain labeled with a green fluorescent protein.
Oxidative Stress Function of the
Saccharomyces cerevisiae
Skn7 Receiver Domain
Authors: He Xin-Jian , Mulford KariAn E. , Fassler Jan S.
Published: March 2009

The bifunctional
Saccharomyces cerevisiae
Skn7 transcription factor regulates osmotic stress response genes as well as oxidative stress response genes; however, the mechanisms involved in these two types of regulation differ. Skn7 osmotic stress activity depends on the phosphorylation of the receiver domain aspartate, D427, by the Sln1 histidine kinase. In contrast, D427 and the SLN1-SKN7 phosphorelay are dispensable for the oxidative stress response, although the receiver domain is required. The majority of oxidative stress response genes regulated by Skn7 also are regulated by the redox-responsive transcription factor Yap1. It is therefore possible that the nuclearly localized Skn7 does not itself respond to the oxidant but simply cooperates with Yap1 when it translocates to the nucleus. We report here that oxidative stress leads to a phosphatase-sensitive, slow-mobility Skn7 variant. This suggests that Skn7 undergoes a posttranslational modification by phosphorylation following exposure to oxidant. Oxidant-dependent Skn7 phosphorylation was eliminated in strains lacking the Yap1 transcription factor. This suggests that the phosphorylation of Skn7 is regulated by Yap1. Mutations in the receiver domain of Skn7 were identified that affect its oxidative stress function. These mutations were found to compromise the association of Yap1 and Skn7 at oxidative stress response gene promoters. A working model is proposed in which the association of Yap1 with Skn7 in the nucleus is a prerequisite for Skn7 phosphorylation and the activation of oxidative stress response genes.
Aspergillus alabamensis
, a New Clinically Relevant Species in the Section
Terrei
Authors: Balajee S. Arunmozhi , Baddley John W. , Peterson Stephen W. , Nickle David , Varga János , Boey Angeline , Lass-Flörl Cornelia , Frisvad Jens C. , Samson Robert A.
Published: March 2009

Phylogenetic analyses of sequences generated from portions of three genes coding for the proteins enolase (
enoA
), β-tubulin (
benA
), and calmodulin (
calM
) of a large number of isolates within the section
Terrei
, genus
Aspergillus
, revealed the presence of a new cryptic species within this section,
Aspergillus alabamensis.
Most members of this new cryptic species were recovered as colonizing isolates from immunocompetent patient populations, had decreased in vitro susceptibilities to the antifungal drug amphotericin B, and were morphologically similar to but genetically distinct from
Aspergillus terreus
isolates.
Silencing of Host CellCYBBGene Expression by the Nuclear Effector AnkA of the Intracellular PathogenAnaplasma phagocytophilum
Authors: Garcia-Garcia Jose C. , Rennoll-Bankert Kristen E. , Pelly Shaaretha , Milstone Aaron M. , Dumler J. Stephen
Published: March 2009

Coevolution of intracellular bacterial pathogens and their host cells resulted in the appearance of effector molecules that when translocated into the host cell modulate its function, facilitating bacterial survival within the hostile host environment. Some of these effectors interact with host chromatin and other nuclear components. In this report, we show that the AnkA protein ofAnaplasma phagocytophilum, which is translocated into the host cell nucleus, interacts with gene regulatory regions of host chromatin and is involved in downregulating expression ofCYBB(gp91phox) and other key host defense genes. AnkA effector protein rapidly accumulated in nuclei of infected cells coincident with changes inCYBBtranscription. AnkA interacted with transcriptional regulatory regions of theCYBBlocus at sites where transcriptional regulators bind. AnkA binding to DNA occurred at regions with high AT contents. Mutation of AT stretches at these sites abrogated AnkA binding. Histone H3 acetylation decreased dramatically at theCYBBlocus duringA. phagocytophiluminfection, particularly around AnkA binding sites. Transcription ofCYBBand other defense genes was significantly decreased in AnkA-transfected HL-60 cells. These data suggest a mechanism by which intracellular pathogens directly regulate host cell gene expression mediated by nuclear effectors and changes in host chromatin structure.
The Biosynthetic Pathway for Myxol-2′ Fucoside (Myxoxanthophyll) in the Cyanobacterium
Synechococcus
sp. Strain PCC 7002
Authors: Graham Joel E. , Bryant Donald A.
Published: March 2009

Synechococcus
sp. strain PCC 7002 produces a variety of carotenoids, which comprise predominantly dicylic β-carotene and two dicyclic xanthophylls, zeaxanthin and synechoxanthin. However, this cyanobacterium also produces a monocyclic myxoxanthophyll, which was identified as myxol-2′ fucoside. Compared to the carotenoid glycosides produced by diverse microorganisms, cyanobacterial myxoxanthophyll and closely related compounds are unusual because they are glycosylated on the 2′-OH rather than on the 1′-OH position of the ψ end of the molecule. In this study, the genes encoding two enzymes that modify the ψ end of myxoxanthophyll in
Synechococcus
sp. strain PCC 7002 were identified. Mutational and biochemical studies showed that open reading frame SynPCC7002_A2032, renamed
cruF
, encodes a 1′-hydroxylase and that open reading frame SynPCC7002_A2031, renamed
cruG
, encodes a 2′-O-glycosyltransferase. The enzymatic activity of CruF was verified by chemical characterization of the carotenoid products synthesized when
cruF
was expressed in a lycopene-producing strain of
Escherichia coli
. Database searches showed that homologs of
cruF
and
cruG
occur in the genomes of all sequenced cyanobacterial strains that are known to produce myxol or the acylic xanthophyll oscillaxanthin. The genomes of many other bacteria that produce hydroxylated carotenoids but do not contain
crtC
homologs also contain
cruF
orthologs. Based upon observable intermediates, a complete biosynthetic pathway for myxoxanthophyll is proposed. This study expands the suite of enzymes available for metabolic engineering of carotenoid biosynthetic pathways for biotechnological applications.
Redundancy of Enzymes for Formaldehyde Detoxification in
Pseudomonas putida
Authors: Roca Amalia , Rodríguez-Herva Jose J. , Ramos Juan L.
Published: March 2009

Pseudomonas putida
KT2440 exhibits redundant formaldehyde dehydrogenases and formate dehydrogenases that contribute to the detoxification of formaldehyde, a highly toxic compound. Physical and transcriptional analyses showed that the open reading frame (ORF) PP0328, encoding one of the formaldehyde dehydrogenases, is self-sufficient, whereas the other functional formaldehyde dehydrogenase gene (ORF PP3970) forms an operon with another gene of unknown function. Two formate dehydrogenase gene clusters (PP0489 to PP0492 and PP2183 to PP2186) were identified, and genes in these clusters were found to form operons. All four transcriptional promoters were mapped by primer extension and revealed the presence of noncanonical promoters expressed at basal level in the exponential growth phase and at a higher level in the stationary phase regardless of the presence of extracellular formaldehyde or formate. These promoters were characterized by a 5′-AG-CCA-C/A-CT-3′ conserved region between −7 and −16. To determine the contribution of the different gene products to formaldehyde and formate mineralization, mutants with single and double mutations of formaldehyde dehydrogenases were generated, and the effect of the mutations on formaldehyde catabolism was tested by measuring
14
CO
2
evolution from
14
C-labeled formaldehyde. The results showed that both enzymes contributed to formaldehyde catabolism. A double mutant lacking these two enzymes still evolved CO
2
from formaldehyde, suggesting the presence of one or more still-unidentified formaldehyde dehydrogenases. Mutants with single and double mutations in the clusters for formate dehydrogenases were also generated, and all of them were able to metabolize [
14
C]formate to
14
CO
2
, suggesting a redundancy of functions that was not limited to only the annotated genes. Single and double mutants deficient in formaldehyde dehydrogenases and formate dehydrogenases exhibited longer lag phases than did the parental strain when confronted with concentrations of formaldehyde close to the MICs. This suggests a role for the detoxification system in tolerance to sublethal concentrations of formaldehyde.
Sialic Acid (
N
-Acetyl Neuraminic Acid) Utilization by
Bacteroides fragilis
Requires a Novel
N
-Acetyl Mannosamine Epimerase
Authors: Brigham Christopher , Caughlan Ruth , Gallegos Rene , Dallas Mary Beth , Godoy Veronica G. , Malamy Michael H.
Published: March 2009

We characterized the
nanLET
operon in
Bacteroides fragilis
, whose products are required for the utilization of the sialic acid
N-
acetyl neuraminic acid (NANA) as a carbon and energy source. The first gene of the operon is
nanL
, which codes for an aldolase that cleaves NANA into
N
-acetyl mannosamine (manNAc) and pyruvate. The next gene,
nanE
, codes for a manNAc/
N
-acetylglucosamine (NAG) epimerase, which, intriguingly, possesses more similarity to eukaryotic renin binding proteins than to other bacterial NanE epimerase proteins. Unphosphorylated manNAc is the substrate of NanE, while ATP is a cofactor in the epimerase reaction. The third gene of the operon is
nanT
, which shows similarity to the major transporter facilitator superfamily and is most likely to be a NANA transporter. Deletion of any of these genes eliminates the ability of
B. fragilis
to grow on NANA. Although
B. fragilis
does not normally grow with manNAc as the sole carbon source, we isolated a
B. fragilis
mutant strain that can grow on this substrate, likely due to a mutation in a NAG transporter; both manNAc transport and NAG transport are affected in this strain. Deletion of the
nanE
epimerase gene or the
rokA
hexokinase gene, whose product phosphorylates NAG, in the manNAc-enabled strain abolishes growth on manNAc. Thus,
B. fragilis
possesses a new pathway of NANA utilization, which we show is also found in other
Bacteroides
species.
Characterization of a Novel Thermostable Carboxylesterase fromGeobacillus kaustophilusHTA426 Shows the Existence of a New Carboxylesterase Family
Authors: Montoro-García Silvia , Martínez-Martínez Irene , Navarro-Fernández José , Takami Hideto , García-Carmona Francisco , Sánchez-Ferrer Álvaro
Published: March 2009

The gene GK3045 (741 bp) fromGeobacillus kaustophilusHTA426 was cloned, sequenced, and overexpressed intoEscherichia coliRosetta (DE3). The deduced protein was a 30-kDa monomeric esterase with high homology to carboxylesterases fromGeobacillus thermoleovoransNY (99% identity) andGeobacillus stearothermophilus(97% identity). This protein suffered a proteolytic cut inE. coli, and the problem was overcome by introducing a mutation in the gene (K212R) without affecting the activity. The resulting Est30 showed remarkable thermostability at 65°C, above the optimum growth temperature ofG. kaustophilusHTA426. The optimum pH of the enzyme was 8.0. In addition, the purified enzyme exhibited stability against denaturing agents, like organic solvents, detergents, and urea. The protein catalyzed the hydrolysis ofp-nitrophenyl esters of different acyl chain lengths, confirming the esterase activity. The sequence analysis showed that the protein contains a catalytic triad formed by Ser93, Asp192, and His222, and the Ser of the active site is located in the conserved motif Gly91-X-Ser93-X-Gly95 included in most esterases and lipases. However, this carboxylesterase showed no more than 17% sequence identity with the closest members in the eight families of microbial carboxylesterases. The three-dimensional structure was modeled by sequence alignment and compared with others carboxylesterases. The topological differences suggested the classification of this enzyme and otherGeobacillus-related carboxylesterases in a new α/β hydrolase family different from IV and VI.
Transsexuality in the Rhizosphere: Quorum Sensing Reversibly Converts
Agrobacterium tumefaciens
from Phenotypically Female to Male
Authors: Cho Hongbaek , Pinto Uelinton M. , Winans Stephen C.
Published: March 2009

Conjugative plasmids generally encode proteins that block the conjugative entry of identical or similar plasmids into the host cell, a phenomenon known as entry exclusion. Here, we demonstrate that two Ti plasmids of
Agrobacterium tumefaciens
encode robust entry exclusion functions. Two proteins, TrbJ and TrbK, can each mediate entry exclusion and act synergistically. The
trbJ
and
trbK
genes are included within the
trb
operon, which is tightly regulated by the quorum-sensing regulator TraR and the cognate acylhomoserine lactone. In the absence of quorum-sensing signals, these proteins are not significantly expressed, and cells lacking TrbJ and TrbK are efficient Ti plasmid recipients. In the presence of these signals, these strains block the entry of Ti plasmids and instead become efficient conjugal donors.
An Orthologue of
Bacteroides fragilis
NanH Is the Principal Sialidase in
Tannerella forsythia
Authors: Thompson Hayley , Homer Karen A. , Rao Susmitha , Booth Veronica , Hosie Arthur H. F.
Published: March 2009

Sialidase activity is a putative virulence factor of the anaerobic periodontal pathogen
Tannerella forsythia
, but it is uncertain which genes encode this activity. Characterization of a putative sialidase, SiaHI, by others, indicated that this protein alone may not be responsible for all of the sialidase activity. We describe a second sialidase in
T. forsythia
(TF0035), an orthologue of
Bacteroides fragilis
NanH, and its expression in
Escherichia coli
. Sialidase activity of the expressed NanH was confirmed by using 2′-(4-methylumbelliferyl)-α-
d
-
N
-acetylneuraminic acid as a substrate. Biochemical characterization of the recombinant
T. forsythia
NanH indicated that it was active over a broad pH range, with optimum activity at pH 5.5. This enzyme has high affinity for 2′-(4-methylumbelliferyl)-α-
d
-
N
-acetylneuraminic acid (

K
m

of 32.9 ± 10.3 μM) and rapidly releases 4-methylumbelliferone (
V
max
of 170.8 ± 11.8 nmol of 4-methylumbelliferone min
−1
mg of protein
−1
).
E. coli
lysates containing recombinant
T. forsythia
NanH cleave sialic acid from a range of substrates, with a preference for α2-3 glycosidic linkages. The genes adjacent to
nanH
encode proteins apparently involved in the metabolism of sialic acid, indicating that the NanH sialidase is likely to be involved in nutrient acquisition.
Biochemical Analysis of a β-
d
-Xylosidase and a Bifunctional Xylanase-Ferulic Acid Esterase from a Xylanolytic Gene Cluster in
Prevotella ruminicola
23
Authors: Dodd Dylan , Kocherginskaya Svetlana A. , Spies M. Ashley , Beery Kyle E. , Abbas Charles A. , Mackie Roderick I. , Cann Isaac K. O.
Published: March 2009

Prevotella ruminicola
23 is an obligate anaerobic bacterium in the phylum
Bacteroidetes
that contributes to hemicellulose utilization within the bovine rumen. To gain insight into the cellular machinery that this organism elaborates to degrade the hemicellulosic polymer xylan, we identified and cloned a gene predicted to encode a bifunctional xylanase-ferulic acid esterase (
xyn10D-fae1A
) and expressed the recombinant protein in
Escherichia coli
. Biochemical analysis of purified Xyn10D-Fae1A revealed that this protein possesses both endo-β-1,4-xylanase and ferulic acid esterase activities. A putative glycoside hydrolase (GH) family 3 β-
d
-glucosidase gene, with a novel PA14-like insertion sequence, was identified two genes downstream of
xyn10D-fae1A
. Biochemical analyses of the purified recombinant protein revealed that the putative β-
d
-glucosidase has activity for
p
NP-β-
d
-xylopyranoside,
p
NP-α-
l
-arabinofuranoside, and xylo-oligosaccharides; thus, the gene was designated
xyl3A
. When incubated in combination with Xyn10D-Fae1A, Xyl3A improved the release of xylose monomers from a hemicellulosic xylan substrate, suggesting that these two enzymes function synergistically to depolymerize xylan. Directed mutagenesis studies of Xyn10D-Fae1A mapped the catalytic sites for the two enzymatic functionalities to distinct regions within the polypeptide sequence. When a mutation was introduced into the putative catalytic site for the xylanase domain (E280S), the ferulic acid esterase activity increased threefold, which suggests that the two catalytic domains for Xyn10D-Fae1A are functionally coupled. Directed mutagenesis of conserved residues for Xyl3A resulted in attenuation of activity, which supports the assignment of Xyl3A as a GH family 3 β-
d
-xylosidase.
Genetic Mapping of Secretion and Functional Determinants of the
Vibrio cholerae
TcpF Colonization Factor
Authors: Krebs Shelly J. , Kirn Thomas J. , Taylor Ronald K.
Published: March 2009

Colonization of the human small intestine by
Vibrio cholerae
requires the type IV toxin-coregulated pilus (TCP). TcpF, which is encoded within the
tcp
operon, is secreted from the bacterial cell by the TCP apparatus and is also essential for colonization. Bacteria lacking
tcpF
are deficient in colonization, and anti-TcpF antibodies are protective in the infant mouse cholera model. In order to elucidate the regions of the protein that are required for secretion through the TCP apparatus and for its function in colonization, random mutagenesis of
tcpF
was performed. Analysis of these mutants suggests that multiple regions throughout the protein influence extracellular secretion and that determinants near the C terminus are important for the function of TcpF in colonization. The TcpF proteins of certain environmental
V. cholerae
isolates with 31% to 66% identity to pathogenic
V. cholerae
TcpF showed higher similarity in regions identified as secretion determinants but diverged in regions found to be important for colonization. These environmental TcpF proteins are secreted from the pathogenic strain; however, they do not mediate colonization in the infant mouse model. Here we provide genetic evidence pointing toward regions of TcpF that influence secretion, as well as regions that play an important role in in vivo colonization.
Identification of a Polymyxin Synthetase Gene Cluster of
Paenibacillus polymyxa
and Heterologous Expression of the Gene in
Bacillus subtilis
Authors: Choi Soo-Keun , Park Soo-Young , Kim Rumi , Kim Seong-Bin , Lee Choong-Hwan , Kim Jihyun F. , Park Seung-Hwan
Published: March 2009

Polymyxin, a long-known peptide antibiotic, has recently been reintroduced in clinical practice because it is sometimes the only available antibiotic for the treatment of multidrug-resistant gram-negative pathogenic bacteria. Lack of information on the biosynthetic genes of polymyxin, however, has limited the study of structure-function relationships and the development of improved polymyxins. During whole genome sequencing of
Paenibacillus polymyxa
E681, a plant growth-promoting rhizobacterium, we identified a gene cluster encoding polymyxin synthetase. Here, we report the complete sequence of the gene cluster and its function in polymyxin biosynthesis. The gene cluster spanning the 40.6-kb region consists of five open reading frames, designated
pmxA
,
pmxB
,
pmxC
,
pmxD
, and
pmxE
. The
pmxC
and
pmxD
genes are similar to genes that encode transport proteins, while
pmxA
,
pmxB
, and
pmxE
encode polymyxin synthetases. The insertional disruption of
pmxE
led to a loss of the ability to produce polymyxin. Introduction of the
pmx
gene cluster into the
amyE
locus of the
Bacillus subtilis
chromosome resulted in the production of polymyxin in the presence of extracellularly added
l
-2,4-diaminobutyric acid. Taken together, our findings demonstrate that the
pmx
gene cluster is responsible for polymyxin biosynthesis.
Surface Location of Individual Residues of SlpA Provides Insight into the
Lactobacillus brevis
S-Layer
Authors: Vilen Heikki , Hynönen Ulla , Badelt-Lichtblau Helga , Ilk Nicola , Jääskeläinen Pentti , Torkkeli Mika , Palva Airi
Published: March 2009

Bacterial surface layer (S-layer) proteins are excellent candidates for in vivo and in vitro nanobiotechnological applications because of their ability to self-assemble into two-dimensional lattices that form the outermost layer of many
Eubacteria
and most
Archaea
species. Despite this potential, knowledge about their molecular architecture is limited. In this study, we investigated SlpA, the S-layer protein of the potentially probiotic bacterium
Lactobacillus brevis
ATCC 8287 by cysteine-scanning mutagenesis and chemical modification. We generated a series of 46 mutant proteins by replacing single amino acids with cysteine, which is not present in the wild-type protein. Most of the replaced amino acids were located in the self-assembly domain (residues 179 to 435) that likely faces the outer surface of the lattice. As revealed by electron microscopy, all the mutant proteins were able to form self-assembly products identical to that of the wild type, proving that this replacement does not dramatically alter the protein conformation. The surface accessibility of the sulfhydryl groups introduced was studied with two maleimide-containing marker molecules, TMM(PEG)
12
(molecular weight [MW], 2,360) and AlexaFluor488-maleimide (MW = 720), using both monomeric proteins in solution and proteins allowed to self-assemble on cell wall fragments. Using the acquired data and available domain information, we assigned the mutated residues into four groups according to their location in the protein monomer and lattice structure: outer surface of the lattice (9 residues), inner surface of the lattice (9), protein interior (12), and protein-protein interface/pore regions (16). This information is essential, e.g., in the development of therapeutic and other health-related applications of
Lactobacillus
S-layers.
The Glutathione/Glutaredoxin System Is Essential for Arsenate Reduction in
Synechocystis
sp. Strain PCC 6803
Authors: López-Maury Luis , Sánchez-Riego Ana María , Reyes José Carlos , Florencio Francisco J.
Published: March 2009

Arsenic resistance in
Synechocystis
sp. strain PCC 6803 is mediated by an operon of three genes in which
arsC
codes for an arsenate reductase with unique characteristics. Here we describe the identification of two additional and nearly identical genes coding for arsenate reductases in
Synechocystis
sp. strain PCC 6803, which we have designed
arsI1
and
arsI2
, and the biochemical characterization of both ArsC (arsenate reductase) and ArsI. Functional analysis of single, double, and triple mutants shows that both ArsI enzymes are active arsenate reductases but that their roles in arsenate resistance are essential only in the absence of ArsC. Based on its biochemical properties, ArsC belongs to a family that, though related to thioredoxin-dependent arsenate reductases, uses the glutathione/glutaredoxin system for reduction, whereas ArsI belongs to the previously known glutaredoxin-dependent family. We have also analyzed the role in arsenate resistance of the three glutaredoxins present in
Synechocystis
sp. strain PCC 6803 both in vitro and in vivo. Only the dithiolic glutaredoxins, GrxA (glutaredoxin A) and GrxB (glutaredoxin B), are able to donate electrons to both types of reductases in vitro, while GrxC (glutaredoxin C), a monothiolic glutaredoxin, is unable to donate electrons to either type. Analysis of glutaredoxin mutant strains revealed that only those lacking the
grxA
gene have impaired arsenic resistance.
Searching for Protein-Protein Interactions within the
Bacillus subtilis
Spore Coat
Authors: Krajčíková Daniela , Lukáčová Magda , Müllerová Denisa , Cutting Simon M. , Barák Imrich
Published: March 2009

The capability of endospores of
Bacillus subtilis
to withstand extreme environmental conditions is secured by several attributes. One of them, the protein shell that encases the spore and is known as the coat, provides the spore with its characteristic resistance to toxic chemicals, lytic enzymes, and predation by unicellular and multicellular eukaryotes. Despite most of the components of the spore coat having been identified, we have only a vague understanding of how such a complex structure is assembled. Using the yeast two-hybrid system, we attempted to identify direct contacts among the proteins allocated to the insoluble fraction of the spore coat: CotV, CotW, CotX, CotY, and CotZ. We also examined whether they could interact with CotE, one of the most crucial morphogenetic proteins governing outer coat formation and also present in the insoluble fraction. Out of all 21 possible interactions we tested, 4 were found to be positive. Among these interactions, we confirmed the previous observation that CotE forms homo-oligomers. In addition, we observed homotypic interactions of CotY, strong interactions between CotZ and CotY, and relatively weak, yet significant, interactions between CotV and CotW. The results of this yeast two-hybrid analysis were confirmed by size exclusion chromatography of recombinant coat proteins and a pull-down assay.
Role of Class A Penicillin-Binding Proteins in the Expression of β-Lactam Resistance in
Enterococcus faecium
Authors: Rice Louis B. , Carias Lenore L. , Rudin Susan , Hutton Rebecca , Marshall Steven , Hassan Medhat , Josseaume Nathalie , Dubost Lionel , Marie Arul , Arthur Michel
Published: March 2009

Peptidoglycan is polymerized by monofunctional
d
,
d
-transpeptidases belonging to class B penicillin-binding proteins (PBPs) and monofunctional glycosyltransferases and by bifunctional enzymes that combine both activities (class A PBPs). Three genes encoding putative class A PBPs (
pbpF, pbpZ
, and
ponA
) were deleted from the chromosome of
Enterococcus faecium
D344R in all possible combinations in order to identify the glycosyltransferases that cooperate with low-affinity class B Pbp5 for synthesis of peptidoglycan in the presence of β-lactam antibiotics. The viability of the triple mutant indicated that glycan strands can be polymerized independently from class A PBPs by an unknown glycosyltranferase. The susceptibility of the Δ
pbpF
Δ
ponA
mutant and triple mutants to extended spectrum cephalosporins (ceftriaxone and cefepime) identified either PbpF or PonA as essential partners of Pbp5 for peptidoglycan polymerization in the presence of the drugs. Mass spectrometry analysis of peptidoglycan structure showed that loss of PonA and PbpF activity led to a minor decrease in the extent of peptidoglycan cross-linking by the remaining PBPs without any detectable compensatory increase in the participation of the
l,d
-transpeptidase in peptidoglycan synthesis. Optical density measurements and electron microscopy analyses showed that the Δ
pbpF
Δ
ponA
mutant underwent increased stationary-phase autolysis compared to the parental strain. Unexpectedly, deletion of the class A
pbp
genes revealed dissociation between the expression of resistance to cephalosporins and penicillins, although the production of Pbp5 was required for resistance to both classes of drugs. Thus, susceptibility of Pbp5-mediated peptidoglycan cross-linking to different β-lactam antibiotics differed as a function of its partner glycosyltransferase.
Three-dimensional reconstruction of sentinel lymph nodes with metastatic breast cancer indicates three distinct patterns of tumour growth
Authors: Paish E C , Green A R , Rakha E A , Macmillan R D , Maddison J R , Ellis I O
Published: March 2009

Aim:

A better understanding of the biology of nodal metastatic disease is of indisputable value. Three-dimensional (3D) serial section alignment and reconstruction techniques can be used for visualisation of nodal metastasis and could provide better understanding of disease growth patterns.

Methods:

19 tumour-involved sentinel nodes (SLNs) from breast cancer patients were serially sectioned, immunohistochemically stained, and digitally scanned. Digital image alignment and voxel-based rendering was used to construct informative 3D visual representations of metastatic tumour distribution within involved nodes.

Results:

The 3D reconstruction technique was successful and informative. The reconstructions of all 19 SLNs enabled the metastatic tumour cells to be viewed infiltrating normal SLN tissue from all angles. Metastases were present at the afferent lymphatic pole in 17/19 cases, confined to the afferent pole only in 7 cases, located at the efferent pole in 12/19 cases, and efferent pole only in just 2 cases. Finally, this study made the novel observation that metastatic growth occurs in three distinct patterns: sinusoidal, nodular and diffuse.

Conclusions:

This methodology provides improved understanding of metastatic disease development and potentially could be used to develop strategies to improve techniques for its routine detection. Further studies are required in order to evaluate the prognostic and biological significance of the growth patterns identified.
Relationships between alumina and bauxite dust exposure and cancer, respiratory and circulatory disease: Table 1
Authors: Friesen M C , Fritschi L , Del Monaco A , Benke G , Dennekamp M , de Klerk N , Hoving J L , MacFarlane E , Sim M R
Published: March 2009

Objectives:

To examine the associations between alumina and bauxite dust exposure and cancer incidence and circulatory and respiratory disease mortality among bauxite miners and alumina refinery workers.

Methods:

This cohort of 5770 males has previously been linked to national mortality and national and state cancer incidence registries (1983–2002). In this paper, Poisson regression was used to undertake internal comparisons within the cohort based on subgroups of cumulative exposure to inhalable bauxite and alumina dust. Exposure was estimated using job histories and historical air monitoring data.

Results:

There was no association between ever bauxite exposure and any of the outcomes. There was a borderline significant association between ever alumina exposure and cerebrovascular disease mortality (10 deaths, RR 3.8, 95% CI 1.1 to 13). There was some evidence of an exposure–response relationship between cumulative bauxite exposure and non-malignant respiratory disease mortality (seven deaths, trend p value: 0.01) and between cumulative alumina exposure and cerebrovascular disease mortality (trend p value: 0.04). These associations were based on very few cases and for non-malignant respiratory disease the deaths represented a heterogeneous mixture of causes. There was no evidence of an excess risk for any cancer type with bauxite or alumina exposure.

Conclusions:

These preliminary findings, based on very few cases, suggest that cumulative inhalable bauxite exposure may be associated with an excess risk of death from non-malignant respiratory disease and that cumulative inhalable alumina dust exposure may be associated with an excess risk of death from cerebrovascular disease. Neither exposure appears to increase the risk of incident cancers.
Txt2stop: a pilot randomised controlled trial of mobile phone-based smoking cessation support
Authors: Free C , Whittaker R , Knight R , Abramsky T , Rodgers A , Roberts I G
Published: March 2009

Aim:

To conduct a pilot randomised controlled trial of mobile phone-based smoking cessation support intervention for the UK population.

Design:

Randomised controlled trial (txt2stop).

Setting:

Community.

Participants:

200 participants responding to radio, poster and leaflet-based promotions regarding the trial.

Main outcome measures:

The response rate for the outcome measures planned for the main trial. Participants’ qualitative responses to open-ended questions about the intervention content. Secondary outcomes were the outcomes planned for the main trial including the point prevalence of self-reported smoking at 4 weeks and pooled effect estimate for the short-term results for the STOMP and txt2stop trials.

Results:

The response rate at 4 weeks was 96% and at 6 months was 92%. The results at 4 weeks show a doubling of self-reported quitting relative risk (RR) 2.08 (95% CI 1.11 to 3.89), 26% vs 12%. The pooled effect estimate combining txt2stop and a previous New Zealand trial in the short term is RR 2.18 (95% CI 1.79 to 2.65).

Conclusions:

Mobile phone-based smoking cessation is an innovative means of delivering smoking cessation support, which doubles the self-reported quit rate in the short term. It could represent an important, but as yet largely unused, medium to deliver age-appropriate public health measures. The long-term effect of this mobile phone-based smoking cessation support will be established by a large randomised controlled trial currently in recruitment.
Comparison between Nurse-Saul and Arrhenius equations
Authors: Jung Jong-Suk , Cho Yoon-Ho
Published: March 2009

The primary objective of the present study is to assess the use of the maturity method to determine the joint sawing window and the traffic opening time on whitetopping construction in Korea. To determine the joint sawing time, it is necessary to determine the minimum strength not to have raveling and to identify the safe time of drying shrinkage. The present study found that the minimum compressive strength for joint sawing was 4.41 MPa (45 kg/cm2) and drying shrinkage occurred just after the concrete temperature peaked. The Nurse-Saul function was used for determining the joint sawing window and the traffic opening time because it better predicted in-place strength. The present study determined that the joint sawing window ranged from 210 °C·h to 310 °C·h (actual time of 6.5 to 9 h) and that the traffic opening time was after maturity values reached 390 °C·h for the mix at 20 °C.
Targeted ablation of cardiac sympathetic neurons reduces resting, reflex and exercise-induced sympathetic activation in conscious rats
Authors: Lujan Heidi L. , Palani Gurunanthan , Chen Ying , Peduzzi Jean D. , DiCarlo Stephen E.
Published: March 2009

Cholera toxin B subunit conjugated to saporin (SAP, a ribosomal inactivating protein that binds to and inactivates ribosomes) was injected in both stellate ganglia to evaluate the physiological response to targeted ablation of cardiac sympathetic neurons. Resting cardiac sympathetic activity (cardiac sympathetic tonus), exercise-induced sympathetic activity (heart rate responses to graded exercise), and reflex sympathetic activity (heart rate responses to graded doses of sodium nitroprusside, SNP) were determined in 18 adult conscious Sprague-Dawley male rats. Rats were randomly divided into the following three groups ( n = 6/group): 1) control (no injection), 2) bilateral stellate ganglia injection of unconjugated cholera toxin B (CTB), and 3) bilateral stellate ganglia injection of cholera toxin B conjugated to SAP (CTB-SAP). CTB-SAP rats, compared with control and CTB rats, had reduced cardiac sympathetic tonus and reduced heart rate responses to graded exercise and graded doses of SNP. Furthermore, the number of stained neurons in the stellate ganglia and spinal cord (segments T1–T4) was reduced in CTB-SAP rats. Thus CTB-SAP retrogradely transported from the stellate ganglia is effective at ablating cardiac sympathetic neurons and reducing resting, exercise, and reflex sympathetic activity. Additional studies are required to further characterize the physiological responses to this procedure as well as determine if this new approach is safe and efficacious for the treatment of conditions associated with excess sympathetic activity (e.g., autonomic dysreflexia, hypertension, heart failure, and ventricular arrhythmias).
Hypoxia during pregnancy in rats leads to early morphological changes of atherosclerosis in adult offspring
Authors: Wang Zhenhua , Huang Ziyang , Lu Guorong , Lin Ling , Ferrari Markus
Published: March 2009

Exposure to an adverse intrauterine environment increases the risk of cardiovascular disease later in adult life. However, the time course relationship between prenatal hypoxia and the onset of atherosclerosis in offspring remains unknown. The purpose of this study is to evaluate the role of reduced fetal oxygen supply on early development of atherogenesis in the adult offspring and further assess its susceptibility to sex-, hyperlipidemia-, and postnatal hypoxemia-related differences. Based on a 4 × 2 full factorial design consisting of four factors of maternal hypoxia, sex, hyperlipidemia, and postnatal hypoxemia, characteristics of growth were determined, and histopathological observation and morphometric analysis of the thoracic aortas were performed in Sprague-Dawley rat offspring. Intrauterine growth restriction, altered body shape at birth, and accelerated postnatal weight gain occurred in the maternal hypoxia group but did not occur in the control group. In 16-mo-old maternal hypoxia offspring, the thoracic aortas exhibited lesions similar to early events in atherosclerosis that involved impaired endothelial cells, thickening and fibration of intimas, infiltration of inflammatory cells to the subendothelial space, and migration and proliferation of vascular smooth muscle cells to the intima. In contrast, no detectable pathological changes were observed in the offspring without maternal hypoxia exposure. Morphometric analysis further demonstrated that prenatal hypoxia caused a significant thickening of intima ( P < 0.001) with a main effect of 5.5 μm, an approximately twofold increase compared with controls. In addition, there was a positive additive relationship between prenatal hypoxia and hyperlipidemia on the intimal thickness ( P < 0.05). There were no other main effects or interaction among these four factors. In summary, our results indicate that maternal hypoxia during pregnancy leads to early pathological appearances of atherogenesis in adult offspring. This effect was enhanced with hyperlipemia but was unaffected by postnatal hypoxia or sex.
Microarray analysis of gene expression in mouse aorta reveals role of the calcium signaling pathway in control of atherosclerosis susceptibility
Authors: Yuan Zuobiao , Miyoshi Toru , Bao Yongde , Sheehan Jason P. , Matsumoto Alan H. , Shi Weibin
Published: March 2009

Inbred mouse strains C57BL/6J (B6) and C3H/HeJ (C3H) exhibit a marked difference in atherosclerotic lesion formation when deficient in apolipoprotein E (apoE−/−), and the arterial wall has been identified as a source of the difference in atherosclerosis susceptibility. In the present study, differences in gene expression in aortic walls of the two strains were analyzed by microarrays. Total RNA was extracted from the aorta of 6-wk-old female B6 and C3H apoE−/− mice fed a chow or Western diet. There were 1,514 genes in chow fed mice and 590 genes in Western fed mice that were found to be differentially expressed between the two strains. Pathway analysis of differentially expressed genes suggested a role for the calcium signaling pathway in regulating atherosclerosis susceptibility. Oxidized LDL (oxLDL) induced a dose-dependent rise in cytosolic calcium levels in B6 endothelial cells. oxLDL-induced monocyte chemoattractant protein-1 production was inhibited by pretreatment with calcium chelator EGTA or intracellular calcium trapping compound BAPTA, indicating that calcium ions mediate the effect of oxLDL on monocyte chemoattractant protein-1 induction. The present findings demonstrate involvement of the calcium signaling pathway in the inflammatory process of atherogenesis.
Thrombospondin-1 modulates VEGF-A-mediated Akt signaling and capillary survival in the developing retina
Authors: Sun Jingfang , Hopkins Benjamin D. , Tsujikawa Kaoru , Perruzzi Carole , Adini Irit , Swerlick Robert , Bornstein Paul , Lawler Jack , Benjamin Laura E.
Published: March 2009

Microvascular development is often perceived to result from a balance of positive and negative factors that impact signaling for proliferation and survival. The survival signaling that results from hypoxia-induced VEGF-A has been well established, but the factors that antagonize this signaling have been poorly studied. As endogenous inhibitors of angiogenesis, thrombospondins (TSPs) are likely candidates to affect survival signaling. Here we report that TSP1 antagonized microvascular survival to retinal hyperoxia, and Akt signaling in both the retina and in cultured endothelial cells. TSP1 expression is correlated with the association of the CD36 receptor with Src versus Fyn. In the presence of TSP1, CD36 is coprecipitated with Fyn as previously shown by others. However, in the absence of TSP1, there is a preferential association with Src. We now demonstrate that these Src family kinases play an important role in modulating microvascular survival in response to TSP1 by crossing tsp1−/−mice to the src−/−and fyn−/−mice and testing the survival of retinal blood vessels in hyperoxia. We find that tsp1−/−, fyn−/−, and double-mutant tsp1−/−/fyn−/−mice have a similar enhancement of capillary survival in oxygen, whereas in a tsp−/−background, the loss of only one allele of src restores the balance in survival and apoptosis to that of wild-type mice. Taken together, we hypothesize that TSP1 antagonizes VEGF-driven Akt survival signaling in part through the recruitment of Fyn to membrane domains containing CD36, but when TSP1 is absent, an opposing Src recruitment contributes to VEGF-driven Akt phosphorylation and capillary survival.