bluuebunny/crossref_metadata_2025 huggingface dataset:
Viral and Flagellate Control of Prokaryotic Production and Community Structure in Offshore Mediterranean Waters
Authors: Bonilla-Findji Osana , Herndl Gerhard J. , Gattuso Jean-Pierre , Weinbauer Markus G.
Published: May 2009

A dilution and size fractionation approach was used to study the separate and combined effects of viruses and flagellates on prokaryotic production ([
3
H]leucine incorporation) and community composition (16S rRNA gene PCR and denaturing gradient gel electrophoresis [DGGE]) in the upper mixed layer and the deep chlorophyll maximum in the offshore Mediterranean Sea. Four experiments were established using differential filtration: a resource control without predators (C treatment), treatment in the presence of viruses (V treatment), treatment in the presence of flagellates (F treatment), and treatment in the presence of both predators (VF treatment). The V and VF treatments increased prokaryotic abundance (1.4- to 2.3-fold) and the number of DGGE bands (by up to 43%) and decreased prokaryotic production compared to the level for the C treatment (by 22 to 99%). For the F treatment, significant differences compared to the level for the C treatment were found as well, but trends were not consistent across experiments. The relative abundances of the high-nucleic-acid subgroups of prokaryotes with high scatter (HNAhs) in flow cytometer settings were lower in the V and VF treatments than in the C and F treatments. These differences were probably due to lysis of very active HNA prokaryotes in the V and VF treatments. Our results indicate that the presence of viruses or viruses plus flagellates sustains prokaryotic diversity and controls prokaryotic production by regulating the proportion of the highly active members of the community. Our data also suggest that lysis and grazing control influences the relationship between bacterial community composition and prokaryotic production.
Community Structure Evolution and Enrichment of Glycogen-Accumulating Organisms Producing Polyhydroxyalkanoates from Fermented Molasses
Authors: Pisco Ana R. , Bengtsson Simon , Werker Alan , Reis Maria A. M. , Lemos Paulo C.
Published: May 2009

An open mixed culture was enriched with glycogen-accumulating organisms (GAOs) by using a sequencing batch reactor and treating an agroindustrial waste (sugar cane molasses) under cyclic anaerobic-aerobic conditions. Over a 1-year operating period, the culture exhibited a very stable GAO phenotype with an average polyhydroxyalkanoate (PHA) content of 17% total suspended solids. However, the GAO microbial community evolved over the course of operation to a culture exhibiting unusual characteristics in producing PHAs comprised of short-chain-length monomers, namely, 3-hydroxybutyrate, 3-hydroxy-2-methylbutyrate, 3-hydroxyvalerate, and 3-hydroxy-2-methylvalerate, and also, up to 31 mol% of the medium-chain-length (MCL) monomer 3-hydroxyhexanoate (3HHx). Microbial community analysis by fluorescence in situ hybridization revealed a concurrent long-term drift in the GAO community balance, from mainly “
Candidatus
Competibacter phosphatis” to mainly
Defluviicoccus vanus
-related organisms. The production of 3HHx was confirmed by
13
C nuclear magnetic resonance (NMR) and appeared to be related to the increased presence of
D. vanus
-related GAOs. These results suggest a broadened spectrum of material, chemical, and mechanical properties that can be achieved for biopolymers produced by open mixed cultures from fermented waste. The increased spectrum of polymer properties brings a wider scope of potential applications.
Free-Living Protozoa in Two Unchlorinated Drinking Water Supplies, Identified by Phylogenic Analysis of 18S rRNA Gene Sequences
Authors: Valster Rinske M. , Wullings Bart A. , Bakker Geo , Smidt Hauke , van der Kooij Dick
Published: May 2009

Free-living protozoan communities in water supplies may include hosts for
Legionella pneumophila
and other undesired bacteria, as well as pathogens. This study aimed at identifying free-living protozoa in two unchlorinated groundwater supplies, using cultivation-independent molecular approaches. For this purpose, samples (<20°C) of treated water, distributed water, and distribution system biofilms were collected from supply A, with a low concentration of natural organic matter (NOM) (<0.5 ppm of C), and from supply B, with a high NOM concentration (7.9 ppm of C). Eukaryotic communities were studied using terminal restriction fragment length polymorphism and clone library analyses of partial 18S rRNA gene fragments and a
Hartmannella vermiformis
-specific quantitative PCR (qPCR). In both supplies, highly diverse eukaryotic communities were observed, including free-living protozoa, fungi, and metazoa. Sequences of protozoa clustered with Amoebozoa (10 operational taxonomic units [OTUs]), Cercozoa (39 OTUs), Choanozoa (26 OTUs), Ciliophora (29 OTUs), Euglenozoa (13 OTUs), Myzozoa (5 OTUs), and Stramenopiles (5 OTUs). A large variety of protozoa were present in both supplies, but the estimated values for protozoan richness did not differ significantly.
H. vermiformis
was observed in both supplies but was not a predominant protozoan. One OTU with the highest similarity to
Acanthamoeba polyphaga
, an opportunistic human pathogen and a host for undesired bacteria, was observed in supply A. The high level of NOM in supply B corresponded with an elevated level of active biomass and with elevated concentrations of
H. vermiformis
in distributed water. Hence, the application of qPCR may be promising in elucidating the relationship between drinking water quality and the presence of specific protozoa.
Rapid Methods for High-Throughput Detection of Sulfoxides
Authors: Shainsky Janna , Derry Netta-Lee , Leichtmann-Bardoogo Yael , Wood Thomas K. , Fishman Ayelet
Published: May 2009

Enantiopure sulfoxides are prevalent in drugs and are useful chiral auxiliaries in organic synthesis. The biocatalytic enantioselective oxidation of prochiral sulfides is a direct and economical approach for the synthesis of optically pure sulfoxides. The selection of suitable biocatalysts requires rapid and reliable high-throughput screening methods. Here we present four different methods for detecting sulfoxides produced via whole-cell biocatalysis, three of which were exploited for high-throughput screening. Fluorescence detection based on the acid activation of omeprazole was utilized for high-throughput screening of mutant libraries of toluene monooxygenases, but no active variants have been discovered yet. The second method is based on the reduction of sulfoxides to sulfides, with the coupled release and measurement of iodine. The availability of solvent-resistant microtiter plates enabled us to modify the method to a high-throughput format. The third method, selective inhibition of horse liver alcohol dehydrogenase, was used to rapidly screen highly active and/or enantioselective variants at position V106 of toluene
ortho
-monooxygenase in a saturation mutagenesis library, using methyl-
p
-tolyl sulfide as the substrate. A success rate of 89% (i.e., 11% false positives) was obtained, and two new mutants were selected. The fourth method is based on the colorimetric detection of adrenochrome, a back-titration procedure which measures the concentration of the periodate-sensitive sulfide. Due to low sensitivity during whole-cell screening, this method was found to be useful only for determining the presence or absence of sulfoxide in the reaction. The methods described in the present work are simple and inexpensive and do not require special equipment.
Cultivation of Methanogens under Low-Hydrogen Conditions by Using the Coculture Method
Authors: Sakai Sanae , Imachi Hiroyuki , Sekiguchi Yuji , Tseng I-Cheng , Ohashi Akiyoshi , Harada Hideki , Kamagata Yoichi
Published: May 2009

We previously reported the isolation of novel methanogens by using a new cultivation method, referred to as the coculture method. Here, we extended our coculture method to various anaerobic environmental samples. As a result, we successfully cultivated some uncharacterized methanogens in coculture enrichments and eventually isolated a new methanogen, within the order
Methanomicrobiales
.
Protein Phosphatase Type 1-Interacting Protein Ysw1 Is Involved in Proper Septin Organization and Prospore Membrane Formation during Sporulation
Authors: Ishihara Makoto , Suda Yasuyuki , Inoue Ichiro , Tanaka Takayuki , Takahashi Tetsuo , Gao Xiao-Dong , Fukui Yasuhisa , Ihara Sayoko , Neiman Aaron M. , Tachikawa Hiroyuki
Published: May 2009

Sporulation of
Saccharomyces cerevisiae
is a developmental process in which four haploid spores are generated inside a diploid cell. Gip1, a sporulation-specific targeting subunit of protein phosphatase type 1, together with its catalytic subunit, Glc7, colocalizes with septins along the extending prospore membrane and is required for septin organization and spore wall formation. However, the mechanism by which Gip1-Glc7 phosphatase promotes these events is unclear. We show here that Ysw1, a sporulation-specific coiled-coil protein, has a functional relationship to Gip1-Glc7 phosphatase. Overexpression of
YSW1
partially suppresses the sporulation defect of a temperature-sensitive allele of
gip1
. Ysw1 interacts with Gip1 in a two-hybrid assay, and this interaction is required for suppression. Ysw1 tagged with green fluorescent protein colocalizes with septins and Gip1 along the extending prospore membrane during spore formation. Sporulation is partially defective in
ysw1
Δ mutant, and cytological analysis revealed that septin structures are perturbed and prospore membrane extension is aberrant in
ysw1
Δ cells. These results suggest that Ysw1 functions with the Gip1-Glc7 phosphatase to promote proper septin organization and prospore membrane formation.
The
Helicobacter pylori
Anti-Sigma Factor FlgM Is Predominantly Cytoplasmic and Cooperates with the Flagellar Basal Body Protein FlhA
Authors: Rust Melanie , Borchert Sophie , Niehus Eike , Kuehne Sarah A. , Gripp Eugenia , Bajceta Afrodita , McMurry Jonathan L. , Suerbaum Sebastian , Hughes Kelly T. , Josenhans Christine
Published: May 2009

Helicobacter pylori
requires flagellar motility and orientation to persist actively in its habitat. A particular feature of flagella in most
Helicobacter
species including
H. pylori
is a membraneous flagellar sheath. The anti-sigma factor FlgM of
H. pylori
is unusual, since it lacks an N-terminal domain present in other FlgM homologs, e.g., FlgM of
Salmonella
spp., whose regulatory function is intimately coupled to its secretion through the flagellar type III secretion system. The aim of the present study was to characterize the localization and secretion of the short
H. pylori
FlgM in the presence of a flagellar sheath and to elucidate its interaction with other flagellar proteins, such as the basal body protein FlhA, which was previously shown to cooperate with FlgM for regulation.
H. pylori
FlgM was only released into the medium in minor amounts in wild-type bacteria, where the bulk amount of the protein was retained in the cytoplasm. Some FlgM was detected in the flagellar fraction. FlgM was expressed in
flhA
mutants and was less soluble and differentially localized in bacterial fractions of the
flhA
mutant in comparison to wild-type bacteria. FlgM-green fluorescent protein and FlgM-V5 translational fusions were generated and expressed in
H. pylori
. FlgM displayed a predominantly polar distribution and interacted with the C-terminal domain of FlhA (FlhA
C
). We suggest that, in
H. pylori
, FlgM secretion may not be paramount for its regulatory function and that protein interactions at the flagellar basal body may determine the turnover and localization of functional FlgM.
Translational Control of the Antibiotic Inducibility of the PA5471 Gene Required for
mexXY
Multidrug Efflux Gene Expression in
Pseudomonas aeruginosa
Authors: Morita Yuji , Gilmour Christie , Metcalf Devon , Poole Keith
Published: May 2009

The PA5471 gene required for induction of the MexXY multidrug efflux system in response to ribosome-targeting antimicrobials was itself shown to be inducible by ribosome-targeting antimicrobials (Y. Morita, M. L. Sobel, and K. Poole, J. Bacteriol. 188:1847-1855, 2006). Using a
lacZ
transcriptional reporter, drug inducibility of PA5471 was shown to require the entirety of the 367-bp PA5472-PA5471 intergenic region. A constitutive promoter activity was, however, localized to the first 75 bp of this region, within which a single PA5471 transcription initiation site was mapped. That 3′ sequences of the intergenic region blocked PA5471 expression and made it antibiotic dependent was suggestive of an attenuation mechanism of control. A 13-amino-acid leader peptide (LP)-encoding open reading frame preceded by a Shine-Dalgarno sequence was identified ca. 250 bp upstream of the PA5471 coding sequence, and its expression and translation were confirmed using a
lacZ
translational reporter. Alteration of the initiation codon (M1T) or introduction of translational stop signals at codons 3 (Q3Am) and 8 (C8Op) of this LP sequence (PA5471.1) yielded high-level constitutive expression of PA5471, suggesting that interference with LP translation was linked to PA5471 gene expression. Consistent with this, a Q3K mutation in the LP sequence maintained the drug inducibility of PA5471 expression. Introduction of the LP Q3Am mutation into the chromosome of
Pseudomonas aeruginosa
yielded stronger expression of PA5471 than did antibiotic (chloramphenicol) exposure of wild-type
P. aeruginosa
, in agreement with
lacZ
transcriptional fusion data. Still, the Q3Am mutation yielded modest expression of
mexXY
, less than that seen for antibiotic-treated wild-type
P. aeruginosa
. These data suggest that PA5471 is not sufficient for MexXY recruitment in response to antibiotic exposure and that additional antibiotic-dependent effects are needed.
Expression of
Kingella kingae
Type IV Pili Is Regulated by σ
54
, PilS, and PilR
Authors: Kehl-Fie Thomas E. , Porsch Eric A. , Miller Sara E. , StGeme Joseph W.
Published: May 2009

Kingella kingae
is a member of the
Neisseriaceae
and is being recognized increasingly as an important cause of serious disease in children. Recent work has demonstrated that
K. kingae
expresses type IV pili that mediate adherence to respiratory epithelial and synovial cells and are selected against during invasive disease. In the current study, we examined the genome of
K. kingae
strain 269-492 and identified homologs of the
rpoN
and the
pilS
and
pilR
genes that are essential for pilus expression in
Pseudomonas aeruginosa
but not in the pathogenic
Neisseria
species. The disruption of either
rpoN
or
pilR
in
K. kingae
resulted in a marked reduction in the level of transcript for the major pilus subunit (
pilA1
) and eliminated piliation. In contrast, the disruption of
pilS
resulted in only partial reduction in the level of
pilA1
transcript and a partial decrease in piliation. Furthermore, the disruption of
pilS
in colony variants with high-density piliation resulted in variants with low-density piliation. Mutations in the promoter region of
pilA1
and gel shift analysis demonstrated that both σ
54
and PilR act directly at the
pilA1
promoter, with PilR binding to two repetitive elements. These data suggest that the regulation of
K. kingae
type IV pilus expression is complex and multilayered, influenced by both the genetic state and environmental cues.
Role of Maltogenic Amylase and Pullulanase in Maltodextrin and Glycogen Metabolism of
Bacillus subtilis
168
Authors: Shim Jae-Hoon , Park Jong-Tae , Hong Jung-Sun , Kim Ki Woo , Kim Myo-Jeong , Auh Jung-Hyuk , Kim Young-Wan , Park Cheon-Seok , Boos Winfried , Kim Jung-Wan , Park Kwan-Hwa
Published: May 2009

The physiological functions of two amylolytic enzymes, a maltogenic amylase (MAase) encoded by
yvdF
and a debranching enzyme (pullulanase) encoded by
amyX
, in the carbohydrate metabolism of
Bacillus subtilis
168 were investigated using
yvdF
,
amyX
, and
yvdF amyX
mutant strains. An immunolocalization study revealed that YvdF was distributed on both sides of the cytoplasmic membrane and in the periplasm during vegetative growth but in the cytoplasm of prespores. Small carbohydrates such as maltoheptaose and β-cyclodextrin (β-CD) taken up by wild-type
B. subtilis
cells via two distinct transporters, the Mdx and Cyc ABC transporters, respectively, were hydrolyzed immediately to form smaller or linear maltodextrins. On the other hand, the
yvdF
mutant exhibited limited degradation of the substrates, indicating that, in the wild type, maltodextrins and β-CD were hydrolyzed by MAase while being taken up by the bacterium. With glycogen and branched β-CDs as substrates, pullulanase showed high-level specificity for the hydrolysis of the outer side chains of glycogen with three to five glucosyl residues. To investigate the roles of MAase and pullulanase in glycogen utilization, the following glycogen-overproducing strains were constructed: a
glg
mutant with a wild-type background,
yvdF glg
and
amyX glg
mutants, and a
glg
mutant with a double mutant (DM) background. The
amyX glg
and
glg
DM strains accumulated significantly larger amounts of glycogen than the
glg
mutant, while the
yvdF glg
strain accumulated an intermediate amount. Glycogen samples from the
amyX glg
and
glg
DM strains exhibited average molecular masses two and three times larger, respectively, than that of glycogen from the
glg
mutant. The results suggested that glycogen breakdown may be a sequential process that involves pullulanase and MAase, whereby pullulanase hydrolyzes the α-1,6-glycosidic linkage at the branch point to release a linear maltooligosaccharide that is then hydrolyzed into maltose and maltotriose by MAase.
Oleate Hydratase Catalyzes the Hydration of a Nonactivated Carbon-Carbon Bond
Authors: Bevers Loes E. , Pinkse Martijn W. H. , Verhaert Peter D. E. M. , Hagen Wilfred R.
Published: May 2009

The hydration of oleic acid into 10-hydroxystearic acid was originally described for a
Pseudomonas
cell extract almost half a century ago. In the intervening years, the enzyme has never been characterized in any detail. We report here the isolation and characterization of oleate hydratase (EC 4.2.1.53) from
Elizabethkingia meningoseptica
.
Characterization of a Gene Cluster Involved in 4-Chlorocatechol Degradation byPseudomonas reinekeiMT1
Authors: Cámara Beatriz , Nikodem Patricia , Bielecki Piotr , Bobadilla Roberto , Junca Howard , Pieper Dietmar H.
Published: May 2009

Pseudomonas reinekeiMT1 has previously been reported to degrade 4- and 5-chlorosalicylate by a pathway with 4-chlorocatechol, 3-chloromuconate, 4-chloromuconolactone, and maleylacetate as intermediates, and a gene cluster channeling various salicylates into an intradiol cleavage route has been reported. We now report that during growth on 5-chlorosalicylate, besides a novel (chloro)catechol 1,2-dioxygenase, C12OccaA, a novel (chloro)muconate cycloisomerase, MCIccaB, which showed features not yet reported, was induced. This cycloisomerase, which was practically inactive with muconate, evolved for the turnover of 3-substituted muconates and transforms 3-chloromuconate into equal amounts ofcis-dienelactone and protoanemonin, suggesting that it is a functional intermediate between chloromuconate cycloisomerases and muconate cycloisomerases. The corresponding genes,ccaA(C12OccaA) andccaB(MCIccaB), were located in a 5.1-kb genomic region clustered with genes encodingtrans-dienelactone hydrolase (ccaC) and maleylacetate reductase (ccaD) and a putative regulatory gene,ccaR, homologous to regulators of the IclR-type family. Thus, this region includes genes sufficient to enable MT1 to transform 4-chlorocatechol to 3-oxoadipate. Phylogenetic analysis showed that C12OccaAand MCIccaBare only distantly related to previously described catechol 1,2-dioxygenases and muconate cycloisomerases. Kinetic analysis indicated that MCIccaBand the previously identified C12OsalD, rather than C12OccaA, are crucial for 5-chlorosalicylate degradation. Thus, MT1 uses enzymes encoded by a completely novel gene cluster for degradation of chlorosalicylates, which, together with a gene cluster encoding enzymes for channeling salicylates into theortho-cleavage pathway, form an effective pathway for 4- and 5-chlorosalicylate mineralization.
Biosynthesis of Aliphatic Polyketides by Type III Polyketide Synthase and Methyltransferase in
Bacillus subtilis
Authors: Nakano Chiaki , Ozawa Hiroki , Akanuma Genki , Funa Nobutaka , Horinouchi Sueharu
Published: May 2009

Type III polyketide synthases (PKSs) synthesize a variety of aromatic polyketides in plants, fungi, and bacteria. The bacterial genome projects predicted that probable type III PKS genes are distributed in a wide variety of gram-positive and -negative bacteria. The gram-positive model microorganism
Bacillus subtilis
contained the
bcsA
-
ypbQ
operon, which appeared to encode a type III PKS and a methyltransferase, respectively. Here, we report the characterization of
bcsA
(renamed
bpsA
, for
Bacillus
pyrone synthase, on the basis of its function) and
ypbQ
, which are involved in the biosynthesis of aliphatic polyketides. In vivo analysis demonstrated that BpsA was a type III PKS catalyzing the synthesis of triketide pyrones from long-chain fatty acyl-coenzyme A (CoA) thioesters as starter substrates and malonyl-CoA as an extender substrate, and YpbQ was a methyltransferase acting on the triketide pyrones to yield alkylpyrone methyl ethers. YpbQ thus was named BpsB because of its functional relatedness to BpsA. In vitro analysis with histidine-tagged BpsA revealed that it used broad starter substrates and produced not only triketide pyrones but also tetraketide pyrones and alkylresorcinols. Although the aliphatic polyketides were expected to localize in the membrane and play some role in modulating the rigidity and properties of the membrane, no detectable phenotypic changes were observed for a
B. subtilis
mutant containing a whole deletion of the
bpsA-bpsB
operon.
Identification of a Catabolite-Responsive Element Necessary for Regulation of the
cry4A
Gene of
Bacillus thuringiensis
subsp.
israelensis
Authors: Kant Sashi , Kapoor Rupam , Banerjee Nirupama
Published: May 2009

Bacillus thuringiensis
subsp.
israelensis
produces a potent mosquitocidal protein, Cry4A. We have identified a 15-bp catabolite responsive element (
cre
), overlapping the −35 element of the
cry4A
promoter. Changing a guanine to adenine at position −49 in the promoter abolished glucose catabolite repression of
cry4A
and enhanced promoter activity two- to threefold. This
cis
regulatory element is essential for controlled toxin synthesis, vital to evolutionary success of
B. thuringiensis
subsp.
israelensis
.
Molecular Characterization and Lytic Activities of
Streptococcus agalactiae
Bacteriophages and Determination of Lysogenic-Strain Features
Authors: Domelier Anne-Sophie , van der Mee-Marquet Nathalie , Sizaret Pierre-Yves , Héry-Arnaud Geneviève , Lartigue Marie-Frédérique , Mereghetti Laurent , Quentin Roland
Published: May 2009

The application of mitomycin C induction to 114 genetically diverse
Streptococcus agalactiae
strains generated 36 phage suspensions. On electron microscopy of the phage suspensions, it was possible to assign the phages to the
Siphoviridae
family, with three different morphotypes (A, B, and C). Phage genetic diversity was evaluated by a PCR-based multilocus typing method targeting key modules located in the packaging, structural, host lysis, lysogeny, replication, and transcriptional regulation clusters and in the integrase genes and by DNA digestion with EcoRI, HindIII, and ClaI. Thirty-three phages clustering in six distantly related molecular phage groups (I to VI) were identified. Each molecular group was morphotype specific except for morphotype A phages, which were found in five of the six phage groups. The various phage groups defined on the basis of molecular group and morphotype had specific lytic activities, suggesting that each recognized particular host cell targets and had particular lytic mechanisms. Comparison of the characteristics of lysogenic and propagating strains showed no difference in the serotype or clonal complex (CC) identified by multilocus sequence typing. However, all the lysogenic CC17 and CC19 strains presented catabolic losses due to a lack of catabolic decay of
dl
-alpha-glycerol-phosphate substrates (CC17) and of alpha-
d
-glucose-1-phosphate (CC19). Moreover, the phages from CC17 lysogenic strains displayed lytic replication in bacterial hosts from all
S. agalactiae
phylogenetic lineages other than CC23, whereas phages obtained from non-CC17 lysogenic strains lysed bacteria of similar evolutionary origin. Our findings suggest that the adaptive evolution of
S. agalactiae
exposed the bacteria of this species to various phage-mediated horizontal gene transfers, which may have affected the fitness of the more virulent clones.
Characterization of γ-Butyrolactone Autoregulatory Signaling Gene Homologs in the Angucyclinone Polyketide WS5995B ProducerStreptomyces acidiscabies
Authors: Healy Frank G. , Eaton Kevin P. , Limsirichai Prajit , Aldrich Joel F. , Plowman Alaina K. , King Russell R.
Published: May 2009

Organisms belonging to the genusStreptomycesproduce numerous important secondary metabolites and undergo a sophisticated morphological differentiation program. In many instances these processes are under the control of γ-butyrolactone (GBL) autoregulatory systems.Streptomyces acidiscabiesstrain 84.104 produces the secondary metabolite aromatic angucyclinone polyketide WS5995B. In order to explore the role of GBL regulatory circuitry in WS5995B production and morphogenesis inS. acidiscabies, a gene cluster encoding GBL autoregulatory signaling homologs was identified and characterized. Two GBL receptor homologs,sabRandsabS, were found flanking a GBL synthase homologsabA. Strains carrying mutations insabSproduced elevated levels of WS5995B and displayed conditional morphological defects reminiscent of defects seen inStreptomyces bldAmutants. Notably,sabSpossesses a TTA codon predicted to be recognized by tRNAleu.sabAmutants produced higher levels of WS5995B than the wild-type strain but to a lesser extent than the levels of WS5995B seen insabSmutants. Purified recombinant SabR and SabS were tested for their abilities to bind predicted AT-rich autoregulatory element (ARE) boxes within thesabRASregion. SabS did not bind any DNA sequences in this region, while SabR bound an ARE box in the region upstream ofsabS. Quantitative reverse transcription-PCR analysis revealed higher levels ofsabStranscript insabRmutants than in the wild-type strain, suggesting thatsabSexpression is repressed by SabR. Based on these data, we propose that theS. acidiscabies sabRASgenes encode components of a signaling pathway which participates in the regulation of WS5995B production and morphogenesis.
Nucleotide Excision Repair Is a Predominant Mechanism for Processing Nitrofurazone-Induced DNA Damage in
Escherichia coli
Authors: Ona Katherine R. , Courcelle Charmain T. , Courcelle Justin
Published: May 2009

Nitrofurazone is reduced by cellular nitroreductases to form
N
2
-deoxyguanine (
N
2
-dG) adducts that are associated with mutagenesis and lethality. Much attention recently has been given to the role that the highly conserved polymerase IV (Pol IV) family of polymerases plays in tolerating adducts induced by nitrofurazone and other
N
2
-dG-generating agents, yet little is known about how nitrofurazone-induced DNA damage is processed by the cell. In this study, we characterized the genetic repair pathways that contribute to survival and mutagenesis in
Escherichia coli
cultures grown in the presence of nitrofurazone. We find that nucleotide excision repair is a primary mechanism for processing damage induced by nitrofurazone. The contribution of translesion synthesis to survival was minor compared to that of nucleotide excision repair and depended upon Pol IV. In addition, survival also depended on both the RecF and RecBCD pathways. We also found that nitrofurazone acts as a direct inhibitor of DNA replication at higher concentrations. We show that the direct inhibition of replication by nitrofurazone occurs independently of DNA damage and is reversible once the nitrofurazone is removed. Previous studies that reported nucleotide excision repair mutants that were fully resistant to nitrofurazone used high concentrations of the drug (200 μM) and short exposure times. We demonstrate here that these conditions inhibit replication but are insufficient in duration to induce significant levels of DNA damage.
Extracytoplasmic Function σ Factors with Overlapping Promoter Specificity Regulate Sublancin Production in
Bacillus subtilis
Authors: Luo Yun , Helmann John D.
Published: May 2009

Bacillus subtilis
harbors seven extracytoplasmic function (ECF) σ factors. At least three ECF σ factors (σ
M
, σ
W
, and σ
X
) are induced by, and provide resistance to, antibiotics and other agents eliciting cell envelope stress. Here, we report that ECF σ factors also contribute to antibiotic production.
B. subtilis
168 strains that are lysogenic for the SPβ bacteriophage produce sublancin, which inhibits the growth of other, nonlysogenic strains. Genetic studies demonstrate that synthesis of sublancin is largely dependent on σ
X
, with a smaller contribution from σ
M
. A
sigM sigX
double mutant is unable to produce sublancin. This defect is primarily due to the fact that the sublancin biosynthesis is positively activated by the transition state regulator and AbrB paralog Abh, which counteracts transcriptional repression of the sublancin biosynthesis operon by AbrB. Ectopic expression of
abh
bypasses the requirement for σ
M
or σ
X
in sublancin synthesis, as does an
abrB
mutation. In addition to their major role in regulating sublancin expression by activating
abh
transcription, σ
X
and σ
M
also have a second role as positive regulators of sublancin expression that is independent of AbrB and Abh. Since sublancin resistance in nonlysogens is largely dependent on σ
W
, ECF σ factors control both sublancin production and resistance.
Biochemical Evidence for ToxR and ToxJ Binding to the
tox
Operons of
Burkholderia glumae
and Mutational Analysis of ToxR
Authors: Kim Jinwoo , Oh Jonghee , Choi Okhee , Kang Yongsung , Kim Hongsup , Goo Eunhye , Ma Jun , Nagamatsu Tomohisa , Moon Jae Sun , Hwang Ingyu
Published: May 2009

Burkholderia glumae
produces toxoflavin, a phytotoxin with a broad host range, which is a key virulence factor in bacterial rice grain rot. Based on genetic analysis, we previously reported that ToxR, a LysR-type regulator, activates both the
toxABCDE
(toxoflavin biosynthesis genes) and
toxFGHI
(toxoflavin transporter genes) operons in the presence of toxoflavin as a coinducer. Quorum sensing regulates the expression of the transcriptional activator ToxJ that is required for
tox
gene expression. Here, we used gel mobility shift and DNase I protection analyses to demonstrate that both ToxR and ToxJ bind simultaneously to the regulatory regions of both
tox
operons. ToxR and ToxJ both bound to the
toxA
and
toxF
regulatory regions, and the sequences for the binding of ToxR to the regulatory regions of both
tox
operons possessed T-N
11
-A motifs. Following random mutagenesis of
toxR
, 10 ToxR mutants were isolated. We constructed a reporter strain, S6K34 (
toxR
′
A
′::Ω
toxF
::Tn
3
-
gusA34
) to evaluate which amino acid residues are important for ToxR activity. Several single amino acid substitutions identified residues that might be important for ToxR binding to DNA and toxoflavin binding. When various toxoflavin derivatives were tested to determine whether toxoflavin is a specific coinducer of ToxR in the S6K34 strain, ToxR, together with toxoflavin, conferred
toxF
expression, whereas 4,8-dihydrotoxoflavin did so only slightly. With these results, we have demonstrated biochemically that
B. glumae
cells control toxoflavin production tightly by the requirement of both ToxJ and toxoflavin as coinducers of ToxR.
Staphylococcal pyrogenic toxins in infant urine samples: a possible marker of transient bacteraemia
Authors: Harrison L M , Morris J A , Lauder R M , Telford D R
Published: May 2009

Aim:

To screen infant urine for staphylococcal pyrogenic toxins as a possible marker for a toxigenic, transient bacteraemia.

Methods:

Nasopharyngeal swabs, skin swabs, stool and urine samples were collected from 30 infants at 2 weeks, 10 weeks and 7 months of age when the infants were healthy, and from infants of 7 months of age when they had a cold. Samples were cultured andStaphylococcusaureusisolates identified. Isolates were tested for the production of staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin C (SEC) and toxic shock syndrome toxin (TSST-1). Urine samples were analysed for the presence of these toxins by ELISA.

Results:

Nasopharyngeal carriage ofS aureusdecreased with age from 50% at 2 weeks of age to 13% in healthy infants at 7 months of age. Carriage was increased in infants over 7 months of age with a cold (36%). Stool carriage remained constant (37–40%) in healthy infants but increased significantly in infants over 7 months of age with a cold (82%). 13.9% of the isolates produced SEB, 16.7% produced SEC and 18% produced TSST-1. Some isolates produced more than one toxin. 43% of infants were colonised at some time with a toxigenicS aureusstrain.S aureustoxins were detected in 9/101 urine samples. The proportion of positive samples was increased with infection and at 10 weeks of age.

Conclusions:

Infants are exposed early in life toS aureuspyrogenic toxins, which can be detected in infant urine samples. Age and infection affect the proportion of positive samples. The pattern of results can be explained by episodes of transient bacteraemia.
High prevalence of autoantibodies in hepatitis A infection: the impact on laboratory profiles
Authors: Moon H W , Noh J K , Hur M , Yun Y M , Lee C H , Kwon S Y
Published: May 2009

Aims:

In the absence of IgM antibodies against hepatitis A virus (HAV), HAV infections can be regarded as autoimmune hepatitis when they show positive autoantibodies by indirect immunofluorescence and lack other viral markers. The aim of this study was to evaluate the prevalence, titres and impact of autoantibodies in Korean patients with HAV infection.

Methods:

The study involved a retrospective review of the electronic medical records of 73 patients with HAV at Konkuk University Hospital from August 2005 to September 2008. The presence and pattern of anti-nuclear antibody, anti-smooth muscle antibody, anti-mitochondrial antibody and anti-liver/kidney microsomal antibody were assessed by indirect immunofluorescence on Hep-2 cells and mouse/kidney sections.

Results:

Of the 73 patients with hepatitis A, 65 (89.0%) showed positive indirect immunofluorescence tests. Of note, most of the positive tests (95.5%) showed a cytoplasmic pattern with filamentous staining of cytoplasmic fibres. There was no significant difference between groups in age or sex. In patients positive for autoantibodies, alanine aminotransferase and leucocyte count were significantly higher, while the increase in globulin was not statistically significant. In terms of titres, globulin was significantly higher in patients with ⩾1:160 titres than in those with ⩽1:80 titres (mean (SD) 3.4 (0.5) versus 2.8 (0.4) g/dl, respectively; p = 0.000).

Conclusions:

The study demonstrated a high prevalence of anticytoplasmic autoantibodies in patients with acute hepatitis A. These data would be useful to aid interpretation of indirect immunofluorescence testing in patients with acute hepatitis, especially in areas with a high prevalence of HAV.
Automobile industry occupations and bladder cancer: a population-based case-control study in southeastern Michigan, USA
Authors: Kobrosly R W , Meliker J R , Nriagu J O
Published: May 2009

Objectives:

To determine whether employees in the automobile industry in Michigan are at elevated risk of urinary bladder cancer.

Methods:

The authors conducted a population-based case-control study including 418 cases and 571 controls. History of employment within the automobile industry was coded according to the US Census Bureau Index of Occupations. Logistic regression analyses were adjusted for age at interview, cigarette smoking status, and highest education level, and used to assess associations between bladder cancer and (1) ever working in particular occupations within the automobile industry; and (2) usual occupation — defined as occupation of longest duration for each subject.

Results:

Ever having worked in the automobile industry and usual employment within the industry exhibited elevated non-significant risks for bladder cancer among assembly line workers, painters and foremen. A higher risk was seen for those who worked for 20 or more years on the assembly line (OR  =  2.10, 95% CI 1.15 to 3.80). Statistical interaction between usual employment on the assembly line and smoking status (>5 pack-years) was demonstrated (OR  =  6.19, 95% CI 2.69 to 14.24).

Conclusions:

Among workers on the assembly line for at least 20 years, we observed an approximately twofold risk for bladder cancer. Heavy smokers working on the assembly line experience a sixfold risk for bladder cancer. Further research is necessary to verify this finding, identify the exposures that might be contributing to bladder cancer on the assembly line, and examine whether those exposures continue to persist in today’s workplace.
Determinants of mobile phone output power in a multinational study: implications for exposure assessment
Authors: Vrijheid M , Mann S , Vecchia P , Wiart J , Taki M , Ardoino L , Armstrong B K , Auvinen A , Bédard D , Berg-Beckhoff G , Brown J , Chetrit A , Collatz-Christensen H , Combalot E , Cook A , Deltour I , Feychting M , Giles G G , Hepworth S J , Hours M , Iavarone I , Johansen C , Krewski D , Kurttio P , Lagorio S , Lönn S , McBride M , Montestrucq L , Parslow R C , Sadetzki S , Schüz J , Tynes T , Woodward A , Cardis E
Published: May 2009

Objectives:

The output power of a mobile phone is directly related to its radiofrequency (RF) electromagnetic field strength, and may theoretically vary substantially in different networks and phone use circumstances due to power control technologies. To improve indices of RF exposure for epidemiological studies, we assessed determinants of mobile phone output power in a multinational study.

Methods:

More than 500 volunteers in 12 countries used Global System for Mobile communications software-modified phones (GSM SMPs) for approximately 1 month each. The SMPs recorded date, time, and duration of each call, and the frequency band and output power at fixed sampling intervals throughout each call. Questionnaires provided information on the typical circumstances of an individual’s phone use. Linear regression models were used to analyse the influence of possible explanatory variables on the average output power and the percentage call time at maximum power for each call.

Results:

Measurements of over 60 000 phone calls showed that the average output power was approximately 50% of the maximum, and that output power varied by a factor of up to 2 to 3 between study centres and network operators. Maximum power was used during a considerable proportion of call time (39% on average). Output power decreased with increasing call duration, but showed little variation in relation to reported frequency of use while in a moving vehicle or inside buildings. Higher output powers for rural compared with urban use of the SMP were observed principally in Sweden where the study covered very sparsely populated areas.

Conclusions:

Average power levels are substantially higher than the minimum levels theoretically achievable in GSM networks. Exposure indices could be improved by accounting for average power levels of different telecommunications systems. There appears to be little value in gathering information on circumstances of phone use other than use in very sparsely populated regions.
Regulation of gap junctional charge selectivity in cells coexpressing connexin 40 and connexin 43
Authors: Heyman Nathanael S. , Kurjiaka David T. , Ek Vitorin Jose F. , Burt Janis M.
Published: May 2009

Expression of connexin 40 (Cx40) and Cx43 in cardiovascular tissues varies as a function of age, injury, and development with unknown consequences on the selectivity of junctional communication and its acute regulation. We investigated the PKC-dependent regulation of charge selectivity in junctions composed of Cx43, Cx40, or both by simultaneous assessment of junctional permeance rate constants (Bdye) for dyes of similar size but opposite charge, N, N, N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl)amino]ethanaminium (NBD-M-TMA; +1) and Alexa 350 (−1). The ratio of dye rate constants (BNBD-M-TMA/BAlexa 350) indicated that Cx40 junctions are cation selective (10.7 ± 0.5), whereas Cx43 junction are nonselective (1.22 ± 0.14). In coexpressing cells, a broad range of junctional selectivities was observed with mean cation selectivity increasing as the Cx40 to Cx43 expression ratio increased. PKC activation reduced or eliminated dye permeability of Cx43 junctions without altering their charge selectivity, had no effect on either permeability or charge selectivity of Cx40 junctions, and significantly increased the cation selectivity of junctions formed by coexpressing cells (approaching charge selectivity of Cx40 junctions). Junctions composed of Cx43 truncated at residue 257 (Cx43tr) were also not charge selective, but when Cx43tr was coexpressed with Cx40, a broad range of junctional selectivities that was unaffected by PKC activation was observed. Thus, whereas the charge selectivities of homomeric/homotypic Cx43 and Cx40 junctions appear invariant, the selectivities of junctions formed by cells coexpressing Cx40 and Cx43 vary considerably, reflecting both their relative expression levels and phosphorylation-dependent regulation. Such regulation could represent a mechanism by which coexpressing cells such as vascular endothelium and atrial cells regulate acutely the selective intercellular communication mediated by their gap junctions.
Acid-sensing ion channels contribute to the metaboreceptor component of the exercise pressor reflex
Authors: McCord Jennifer L. , Tsuchimochi Hirotsugu , Kaufman Marc P.
Published: May 2009

The exercise pressor reflex is evoked by both mechanical and metabolic stimuli arising in contracting skeletal muscle. Recently, the blockade of acid-sensing ion channels (ASICs) with amiloride and A-316567 attenuated the reflex. Moreover, amiloride had no effect on the mechanoreceptor component of the reflex, prompting us to determine whether ASICs contributed to the metaboreceptor component of the exercise pressor reflex. The metaboreceptor component can be assessed by measuring mean arterial pressure during postcontraction circulatory occlusion when only the metaboreceptors are stimulated. We examined the effects of amiloride (0.5 μg/kg), A-317567 (10 mM, 0.5 ml), and saline (0.5 ml) on the pressor response to and after static contraction while the circulation was occluded in 30 decerebrated cats. Amiloride ( n = 11) and A-317567 ( n = 7), injected into the arterial supply of the triceps surae muscles, attenuated the pressor responses both to contraction while the circulation was occluded and to postcontraction circulatory occlusion (all, P < 0.05). Saline ( n = 11), however, had no effect on the pressor responses to contraction while the circulation was occluded or to postcontraction circulatory occlusion (both, P > 0.79). Our findings led us to conclude that ASICs contribute to the metaboreceptor component of the exercise pressor reflex.
Interstitial flow promotes vascular fibroblast, myofibroblast, and smooth muscle cell motility in 3-D collagen I via upregulation of MMP-1
Authors: Shi Zhong-Dong , Ji Xin-Ying , Qazi Henry , Tarbell John M.
Published: May 2009

Neointima formation often occurs in regions where the endothelium has been damaged and the transmural interstitial flow is elevated. Vascular smooth muscle cells (SMCs) and fibroblasts/myofibroblasts (FBs/MFBs) contribute to intimal thickening by migrating from the media and adventitia into the site of injury. In this study, for the first time, the direct effects of interstitial flow on SMC and FB/MFB migration were investigated in an in vitro three-dimensional system. Collagen I gels were used to mimic three-dimensional extracellular matrix (ECM) for rat aortic SMCs and FBs/MFBs. Exposure to interstitial flow induced by 1 cmH2O pressure differential (shear stress, ∼0.05 dyn/cm2; flow velocity, ∼0.5 μm/s; and Darcy permeability, ∼10−11 cm2) substantially enhanced cell motility. Matrix metalloproteinase (MMP) inhibitor (GM-6001) abolished flow-induced migration augmentation, which suggested that the enhanced motility was MMP dependent. The upregulation of MMP-1 played a critical role for the flow-enhanced motility, which was further confirmed by silencing MMP-1 gene expression. Longer exposures to higher flows suppressed the number of migrated cells, although MMP-1 gene expression remained high. This suppression was a result of both flow-induced tissue inhibitor of metalloproteinase-1 upregulation and increased apoptotic and necrotic cell death. Interstitial flow did not affect MMP-2 gene expression or activity in the collagen I gel for any cell type. Our findings shed light on the mechanism by which vascular SMCs and FBs/MFBs contribute to intimal thickening in regions of vascular injury where interstitial flow is elevated.
Vascular endothelial dysfunction with aging: endothelin-1 and endothelial nitric oxide synthase
Authors: Donato Anthony J. , Gano Lindsey B. , Eskurza Iratxe , Silver Annemarie E. , Gates Phillip E. , Jablonski Kristen , Seals Douglas R.
Published: May 2009

To determine whether impaired endothelium-dependent dilation (EDD) in older adults is associated with changes in the expression of major vasoconstrictor or vasodilator proteins in the vascular endothelium, endothelial cells (EC) were obtained from the brachial artery and peripheral veins of 56 healthy men, aged 18–78 yr. Brachial artery EC endothelin-1 (ET-1) [0.99 ± 0.10 vs. 0.57 ± 0.10 ET-1/human umbilical vein EC (HUVEC) intensity, P = 0.01] and serine 1177 phosphorylated endothelial nitric oxide synthase (PeNOS) (0.77 ± 0.09 vs. 0.44 ± 0.07 PeNOS/HUVEC intensity, P < 0.05) (quantitative immunofluorescence) were greater, and EDD (peak forearm blood flow to intrabrachial acetylcholine) was lower (10.2 ± 0.9 vs. 14.7 ± 1.7 ml·100 ml−1·min−1, P < 0.05) in older ( n = 18, 62 ± 1 yr) vs. young ( n = 15, 21 ± 1 yr) healthy men. EDD was inversely related to expression of ET-1 ( r = −0.39, P < 0.05). Brachial artery EC eNOS expression did not differ significantly with age, but tended to be greater in the older men (young: 0.23 ± 0.03 vs. older: 0.33 ± 0.07 eNOS/HUVEC intensity, P = 0.08). In the sample with venous EC collections, EDD (brachial artery flow-mediated dilation) was lower (3.50 ± 0.44 vs. 7.68 ± 0.43%, P < 0.001), EC ET-1 and PeNOS were greater ( P < 0.05), and EC eNOS was not different in older ( n = 23, 62 ± 1 yr) vs. young ( n = 27, 22 ± 1 yr) men. EDD was inversely related to venous EC ET-1 ( r = −0.37, P < 0.05). ET-1 receptor A inhibition with BQ-123 restored 60% of the age-related impairment in carotid artery dilation to acetylcholine in B6D2F1 mice (5–7 mo, n = 8; 30 mo, n = 11; P < 0.05). ET-1 expression is increased in vascular EC of healthy older men and is related to reduced EDD, whereas ET-1 receptor A signaling tonically suppresses EDD in old mice. Neither eNOS nor PeNOS is reduced with aging. Changes in ET-1 expression and bioactivity, but not eNOS, contribute to vascular endothelial dysfunction with aging.