bluuebunny/crossref_metadata_2025 huggingface dataset:
Integrated Bioprocessing for the pH-Dependent Production of 4-Valerolactone from Levulinate in
Pseudomonas putida
KT2440
Authors: Martin Collin H. , Wu Danyi , Prather Kristala L. Jones
Published: November 2009

Enzymes are powerful biocatalysts capable of performing specific chemical transformations under mild conditions, yet as catalysts they remain subject to the laws of thermodynamics, namely, that they cannot catalyze chemical reactions beyond equilibrium. Here we report the phenomenon and application of using extracytosolic enzymes and medium conditions, such as pH, to catalyze metabolic pathways beyond their intracellular catalytic limitations. This methodology, termed “integrated bioprocessing” because it integrates intracellular and extracytosolic catalysis, was applied to a lactonization reaction in
Pseudomonas putida
for the economical and high-titer biosynthesis of 4-valerolactone from the inexpensive and renewable source levulinic acid. Mutant paraoxonase I (PON1) was expressed in
P. putida
, shown to export from the cytosol in
Escherichia coli
and
P. putida
using an N-terminal sequence, and demonstrated to catalyze the extracytosolic and pH-dependent lactonization of 4-hydroxyvalerate to 4-valerolactone. With this production system, the titer of 4-valerolactone was enhanced substantially in acidic medium using extracytosolically expressed lactonase versus an intracellular lactonase: from <0.2 g liter
−1
to 2.1 ± 0.4 g liter
−1
at the shake flask scale. Based on these results, the production of 4-hydroxyvalerate and 4-valerolactone was examined in a 2-liter bioreactor, and titers of 27.1 g liter
−1
and 8.2 g liter
−1
for the two respective compounds were achieved. These results illustrate the utility of integrated bioprocessing as a strategy for enabling production from novel metabolic pathways and enhancing product titers.
Elimination of Glycerol Production in Anaerobic Cultures of a
Saccharomyces cerevisiae
Strain Engineered To Use Acetic Acid as an Electron Acceptor
Authors: Medina Víctor Guadalupe , Almering Marinka J. H. , van Maris Antonius J. A. , Pronk Jack T.
Published: November 2009

In anaerobic cultures of wild-type
Saccharomyces cerevisiae
, glycerol production is essential to reoxidize NADH produced in biosynthetic processes. Consequently, glycerol is a major by-product during anaerobic production of ethanol by
S. cerevisiae
, the single largest fermentation process in industrial biotechnology. The present study investigates the possibility of completely eliminating glycerol production by engineering
S. cerevisiae
such that it can reoxidize NADH by the reduction of acetic acid to ethanol via NADH-dependent reactions. Acetic acid is available at significant amounts in lignocellulosic hydrolysates of agricultural residues. Consistent with earlier studies, deletion of the two genes encoding NAD-dependent glycerol-3-phosphate dehydrogenase (
GPD1
and
GPD2
) led to elimination of glycerol production and an inability to grow anaerobically. However, when the
E. coli mhpF
gene, encoding the acetylating NAD-dependent acetaldehyde dehydrogenase (EC 1.2.1.10; acetaldehyde + NAD
+
+ coenzyme A ↔ acetyl coenzyme A + NADH + H
+
), was expressed in the
gpd1
Δ
gpd2
Δ strain, anaerobic growth was restored by supplementation with 2.0 g liter
−1
acetic acid. The stoichiometry of acetate consumption and growth was consistent with the complete replacement of glycerol formation by acetate reduction to ethanol as the mechanism for NADH reoxidation. This study provides a proof of principle for the potential of this metabolic engineering strategy to improve ethanol yields, eliminate glycerol production, and partially convert acetate, which is a well-known inhibitor of yeast performance in lignocellulosic hydrolysates, to ethanol. Further research should address the kinetic aspects of acetate reduction and the effect of the elimination of glycerol production on cellular robustness (e.g., osmotolerance).
Expression of the
Clostridium botulinum
A2 Neurotoxin Gene Cluster Proteins and Characterization of the A2 Complex
Authors: Lin Guangyun , Tepp William H. , Pier Christina L. , Jacobson Mark J. , Johnson Eric A.
Published: November 2009

Clostridium botulinum
subtype A2 possesses a botulinum neurotoxin type A (BoNT/A) gene cluster consisting of an
orfX
cluster containing open reading frames (ORFs) of unknown functions. To better understand the association between the BoNT/A2 complex proteins, first, the
orfX
cluster proteins (ORFX1, ORFX3, P47, and the middle part of NTNH) from
C. botulinum
A2 strain Kyoto F and NTNH of A1 strain ATCC 3502 were expressed by using either an
Escherichia coli
or a
C. botulinum
expression system. Polyclonal antibodies against individual
orfX
cluster proteins were prepared by immunizing a rabbit and mice against the expressed proteins. Antibodies were then utilized as probes to determine which of the A2
orfX
cluster genes were expressed in the native A2 culture. N-terminal protein sequencing was also employed to specifically detect ORFX2. Results showed that all of the neurotoxin cluster proteins, except ORFX1, were expressed in the A2 culture. A BoNT/A2 toxin complex (TC) was purified which showed that
C. botulinum
A2 formed a medium-size (300-kDa) TC composed of BoNT/A2 and NTNH without any of the other OrfX cluster proteins. NTNH subtype-specific immunoreactivity was also discovered, allowing for the differentiation of subtypes based on cluster proteins associated with BoNT.
Role of Extracellular Polymeric Substances in the Surface Chemical Reactivity of
Hymenobacter aerophilus
, a Psychrotolerant Bacterium
Authors: Baker M. G. , Lalonde S. V. , Konhauser K. O. , Foght J. M.
Published: November 2009

Bacterial surface layers, such as extracellular polymeric substances (EPS), are known to play an important role in metal sorption and biomineralization; however, there have been very few studies investigating how environmentally induced changes in EPS production affect the cell's surface chemistry and reactivity. Acid-base titrations, cadmium adsorption assays, and Fourier transform infrared spectroscopy (FT-IR) were used to characterize the surface reactivities of
Hymenobacter aerophilus
cells with intact EPS (WC) or stripped of EPS (SC) and purified EPS alone. Linear programming modeling of titration data showed SC to possess functional groups corresponding to phosphoryl (pK
a
∼6.5), phosphoryl/amine (pK
a
∼7.9), and amine/hydroxyl (pK
a
∼9.9). EPS and WC both possess carboxyl groups (pK
a
∼5.1 to 5.8) in addition to phosphoryl and amine groups. FT-IR confirmed the presence of polysaccharides and protein in purified EPS that can account for the additional carboxyl groups. An increased ligand density was observed for WC relative to that for SC, leading to an increase in the amount of Cd adsorbed (0.53 to 1.73 mmol/liter per g [dry weight] and 0.53 to 0.59 mmol/liter per g [dry weight], respectively). Overall, the presence of EPS corresponds to an increase in the number and type of functional groups on the surface of
H. aerophilus
that is reflected by increased metal adsorption relative to that for EPS-free cells.
Quantification of Cyprinid Herpesvirus 3 in Environmental Water by Using an External Standard Virus
Authors: Honjo Mie N. , Minamoto Toshifumi , Matsui Kazuaki , Uchii Kimiko , Yamanaka Hiroki , Suzuki Alata A. , Kohmatsu Yukihiro , Iida Takaji , Kawabata Zen'ichiro
Published: November 2009

Cyprinid herpesvirus 3 (CyHV-3), a lethal DNA virus that spreads in natural lakes and rivers, infects common carp and koi. We established a quantification method for CyHV-3 that includes a viral concentration method and quantitative PCR combined with an external standard virus. Viral concentration methods were compared using the cation-coated filter and ultrafiltration methods. The recovery of virus-like particles was similar for the two methods (cation-coated filter method, 44% ± 19%,n= 3; ultrafiltration method, 50% ± 3%,n= 3); however, the former method was faster and more suitable for routine determinations. The recovery of seeded CyHV-3 based on the cation-coated filter method varied by more than 3 orders of magnitude among the water samples. The recovery yield of CyHV-3 was significantly correlated with that of the seeded λ phage, and the average ratio of λ to the CyHV-3 recovery yield was 1.4, indicating that λ is useful as an external standard virus for determining the recovery yield of CyHV-3. Therefore, to quantify CyHV-3 in environmental water, a known amount of λ was added as an external standard virus to each water sample. Using this method, CyHV-3 DNA was detected in 6 of the 10 (60%) types of environmental water tested; the highest concentration of CyHV-3 DNA was 2 × 105copies liter−1. The lowest recovery limit of CyHV-3 DNA was 60 copies liter−1. This method is practical for monitoring CyHV-3 abundance in environmental water.
Tertiary Structure and Characterization of a Glycoside Hydrolase Family 44 Endoglucanase from
Clostridium acetobutylicum
Authors: Warner Christopher D. , Hoy Julie A. , Shilling Taran C. , Linnen Michael J. , Ginder Nathaniel D. , Ford Clark F. , Honzatko Richard B. , Reilly Peter J.
Published: November 2009

A gene encoding a glycoside hydrolase family 44 (GH44) protein from
Clostridium acetobutylicum
ATCC 824 was synthesized and transformed into
Escherichia coli.
The previously uncharacterized protein was expressed with a C-terminal His tag and purified by nickel-nitrilotriacetic acid affinity chromatography. Crystallization and X-ray diffraction to a 2.2-Å resolution revealed a triose phosphate isomerase (TIM) barrel-like structure with additional Greek key and β-sandwich folds, similar to other GH44 crystal structures. The enzyme hydrolyzes cellotetraose and larger cellooligosaccharides, yielding an unbalanced product distribution, including some glucose. It attacks carboxymethylcellulose and xylan at approximately the same rates. Its activity on carboxymethylcellulose is much higher than that of the isolated
C. acetobutylicum
cellulosome. It also extensively converts lichenan to oligosaccharides of intermediate size and attacks Avicel to a limited extent. The enzyme has an optimal temperature in a 10-min assay of 55°C and an optimal pH of 5.0.
Traceability of Marketable Japanese Shoro in New Zealand: Using Multiplex PCR To Exploit Phylogeographic Variation among Taxa in the
Rhizopogon
Subgenus
Roseoli
Authors: Visnovsky Sandra B. , Guerin-Laguette Alexis , Wang Yun , Pitman Andrew R.
Published: November 2009

Rhizopogon roseolus
Corda (synonym
Rhizopogon rubescens
Tul.), an economically important edible mushroom associated with the Pinaceae (mostly
Pinus
sp.), has a global distribution resulting from the introduction of exotic trees into the Southern Hemisphere for plantation forestry. However, the marketability of
R. roseolus
varies with the place of origin.
R. roseolus
strains cultivated in New Zealand from local carpophores for the Japanese market are morphologically and biologically distinct from those produced in Japan and are consequently considered less valuable. In this study, the ITS1-5.8S-ITS2 rRNA (internal transcribed spacer [ITS]) region was used to examine the phylogenetic relationships of
R. roseolus
and other closely related fungi belonging to
Rhizopogon
subgenus
Roseoli
to determine the genetic basis for phenotypic differences among
R. roseolus
isolates from different geographic regions. Phylogenetic comparison revealed phylogeographic variation within
Rhizopogon
subgenus
Roseoli
. Collections from the United States and Europe grouped into four distinct clades.
Rhizopogon roseolus
isolates found in New Zealand were closely related to those from the United States, likely due to introduction of
Pinus radiata
from its native California in the United States. In contrast, Japanese
R. roseolus
isolates clustered closely with European collections. Phylogenetic differences between Japanese and New Zealand
R. roseolus
isolates may explain the morphological and biological properties attributed to these geographical variants. The ITS region was subsequently used to design a multiplex PCR for the simultaneous identification of Japanese and New Zealand
R. roseolus
isolates to track the establishment of ectomycorrhiza on
P. radiata
seedlings inoculated with commercially valuable
R. roseolus
. This diagnostic demonstrated the first fruiting of Japanese shoro cultivated on
P. radiata
in the Southern Hemisphere.
Identification of theGeobacter metallireducensBamVW Two-Component System, Involved in Transcriptional Regulation of Aromatic Degradation
Authors: Juárez Javier F. , Zamarro María Teresa , Barragán María J. L. , Blázquez Blas , Boll Matthias , Kuntze Kevin , García José Luis , Díaz Eduardo , Carmona Manuel
Published: November 2009

Regulation of aromatic degradation in obligate anaerobes was studied in the Fe(III)-respiring model organismGeobacter metallireducensGS-15. A two-component system and a σ54-dependent promoter were identified that are both involved in the regulation of the gene coding for benzoate-coenzyme A ligase, catalyzing the initial step of benzoate degradation.
The Malaria Parasite Cyclic GMP-Dependent Protein Kinase Plays a Central Role in Blood-Stage Schizogony
Authors: Taylor Helen M. , McRobert Louisa , Grainger Munira , Sicard Audrey , Dluzewski Anton R. , Hopp Christine S. , Holder Anthony A. , Baker David A.
Published: November 2009

A role for the
Plasmodium falciparum
cyclic GMP (cGMP)-dependent protein kinase (PfPKG) in gametogenesis in the malaria parasite was elucidated previously. In the present study we examined the role of PfPKG in the asexual blood-stage of the parasite life cycle, the stage that causes malaria pathology. A specific PKG inhibitor (compound 1, a trisubstituted pyrrole) prevented the progression of
P. falciparum
schizonts through to ring stages in erythrocyte invasion assays. Addition of compound 1 to ring-stage parasites allowed normal development up to 30 h postinvasion, and segmented schizonts were able to form. However, synchronized schizonts treated with compound 1 for ≥6 h became large and dysmorphic and were unable to rupture or liberate merozoites. To conclusively demonstrate that the effect of compound 1 on schizogony was due to its selective action on PfPKG, we utilized genetically manipulated
P. falciparum
parasites expressing a compound 1-insensitive PfPKG. The mutant parasites were able to complete schizogony in the presence of compound 1 but not in the presence of the broad-spectrum protein kinase inhibitor staurosporine. This shows that PfPKG is the primary target of compound 1 during schizogony and provides direct evidence of a role for PfPKG in this process. Discovery of essential roles for the
P. falciparum
PKG in both asexual and sexual development demonstrates that cGMP signaling is a key regulator of both of these crucial life cycle phases and defines this molecule as an exciting potential drug target for both therapeutic and transmission blocking action against malaria.
Evolutionary Dynamics of Mating-Type Loci of
Mycosphaerella
spp. Occurring on Banana
Authors: Arzanlou Mahdi , Crous Pedro W. , Zwiers Lute-Harm
Published: November 2009

The devastating Sigatoka disease complex of banana is primarily caused by three closely related heterothallic fungi belonging to the genus
Mycosphaerella
:
M. fijiensis
,
M. musicola
, and
M. eumusae
. Previous phylogenetic work showing common ancestry led us to analyze the mating-type loci of these
Mycosphaerella
species occurring on banana. We reasoned that this might provide better insight into the evolutionary history of these species. PCR and chromosome-walking approaches were used to clone the mating-type loci of
M. musicola
and
M. eumusae
. Sequences were compared to the published mating-type loci of
M. fijiensis
and other
Mycosphaerella
spp., and a novel organization of the MAT loci was found. The mating-type loci of the examined
Mycosphaerella
species are expanded, containing two additional
Mycosphaerella
-specific genes in a unique genomic organization. The proteins encoded by these novel genes show a higher interspecies than intraspecies homology. Moreover,
M. fijiensis
,
M. musicola
, and
M. eumusae
contain two additional mating-type-like loci, containing parts of both
MAT
1
-
1
-
1
and
MAT
1
-
2
-
1
. The data indicate that
M. fijiensis
,
M. musicola
, and
M. eumusae
share an ancestor in which a fusion event occurred between
MAT
1
-
1
-
1
and
MAT
1
-
2
-
1
sequences and in which additional genes became incorporated into the idiomorph. The new genes incorporated have since then evolved independently in the MAT1-1 and MAT1-2 loci. Thus, these data are an example of the evolutionary dynamics of fungal MAT loci in general and show the great flexibility of the MAT loci of
Mycosphaerella
species in particular.
RNA Interference Silencing of a Major Lipid Droplet Protein Affects Lipid Droplet Size in
Chlamydomonas reinhardtii
Authors: Moellering Eric R. , Benning Christoph
Published: November 2009

Eukaryotic cells store oils in the chemical form of triacylglycerols in distinct organelles, often called lipid droplets. These dynamic storage compartments have been intensely studied in the context of human health and also in plants as a source of vegetable oils for human consumption and for chemical or biofuel feedstocks. Many microalgae accumulate oils, particularly under conditions limiting to growth, and thus have gained renewed attention as a potentially sustainable feedstock for biofuel production. However, little is currently known at the cellular or molecular levels with regard to oil accumulation in microalgae, and the structural proteins and enzymes involved in the biogenesis, maintenance, and degradation of algal oil storage compartments are not well studied. Focusing on the model green alga
Chlamydomonas reinhardtii
, the accumulation of triacylglycerols and the formation of lipid droplets during nitrogen deprivation were investigated. Mass spectrometry identified 259 proteins in a lipid droplet-enriched fraction, among them a major protein, tentatively designated major lipid droplet protein (MLDP). This protein is specific to the green algal lineage of photosynthetic organisms. Repression of MLDP gene expression using an RNA interference approach led to increased lipid droplet size, but no change in triacylglycerol content or metabolism was observed.
Identification and Partial Characterization of a Dynamin-Like Protein, EhDLP1, from the Protist Parasite
Entamoeba histolytica
Authors: Jain Ruchi , Shrimal Shiteshu , Bhattacharya Sudha , Bhattacharya Alok
Published: November 2009

The dynamin superfamily of proteins includes a large repertoire of evolutionarily conserved GTPases that interact with different subcellular organelle membranes in eukaryotes. Dynamins are thought to participate in a number of cellular processes involving membrane remodeling and scission. Dynamin-like proteins (DLPs) form a subfamily of this vast class and play important roles in cellular processes, such as mitochondrial fission, cytokinesis, and endocytosis. In the present study, a gene encoding a dynamin-like protein (EhDLP1) from the protist parasite
Entamoeba histolytica
was identified and the protein was partially characterized using a combination of
in silico
, biochemical, and imaging methods. The protein was capable of GTP binding and hydrolysis, lipid binding, and oligomerization. Immunofluorescence studies showed the protein to be associated with the nuclear membrane. A mutant of EhDLP1 lacking GTP binding and hydrolyzing activities did not associate with the nuclear membrane. The results suggest a nucleus-associated function for EhDLP1.
abc3
+
Encodes an Iron-Regulated Vacuolar ABC-Type Transporter in
Schizosaccharomyces pombe
Authors: Pouliot Benoît , Jbel Mehdi , Mercier Alexandre , Labbé Simon
Published: November 2009

Studies have shown the fundamental contribution of the yeast vacuole as a site for storage and detoxification of metals. Whereas the transmembrane proteins responsible for iron transport into and out of the vacuole have been identified in
Saccharomyces cerevisiae
, less information is available concerning the mobilization of vacuolar iron stores in
Schizosaccharomyces pombe
. In this study, we report the identification of a gene designated
abc3

+

that encodes a protein which exhibits sequence homology with the ABCC subfamily of ATP-binding cassette transporters. The transcription of
abc3

+

is induced by low concentrations of iron but repressed by high levels of iron. The iron-mediated repression of
abc3

+

required a functional
fep1

+

gene. Chromatin immunoprecipitation assays showed that Fep1 associates with the
abc3

+

promoter in vivo, in an iron-dependent manner. Microscopic analyses revealed that a functional Abc3-green fluorescent protein localizes to the membrane vacuole when iron levels were low. Abc3 was required for growth in low-iron medium in the absence of the transport system mediated by Fio1 and Fip1.
abc3Δ
cells exhibited increased levels of expression of the
frp1

+

-encoded ferric reductase, suggesting a loss of Fep1 repression and, consequently, the activation of Fep1-regulated genes. When
abc3

+

was expressed using the
nmt1

+

promoter system, its induction led to a reduced transcriptional activity of the
frp1

+

gene. Because
S. pombe
does not possess vacuolar membrane-localized orthologs to
S. cerevisiae
Fth1, Fet5, and Smf3, our findings suggested that Abc3 may be responsible for mobilizing stored iron from the vacuole to the cytosol in response to iron deficiency.
Nucleosomes Are Depleted at the
VSG
Expression Site Transcribed by RNA Polymerase I in African Trypanosomes
Authors: Figueiredo Luisa M. , Cross George A. M.
Published: November 2009

In most eukaryotes, RNA polymerase I (Pol I) exclusively transcribes long arrays of identical rRNA genes (ribosomal DNA [rDNA]). African trypanosomes have the unique property of using Pol I to also transcribe the variant surface glycoprotein
VSG
genes. VSGs are important virulence factors because their switching allows trypanosomes to escape the host immune system, a mechanism known as antigenic variation. Only one
VSG
is transcribed at a time from one of 15 bloodstream-form expression sites (BESs). Although it is clear that switching among BESs does not involve DNA rearrangements and that regulation is probably epigenetic, it remains unknown why BESs are transcribed by Pol I and what roles are played by chromatin structure and histone modifications. Using chromatin immunoprecipitation, micrococcal nuclease digestion, and chromatin fractionation, we observed that there are fewer nucleosomes at the active BES and that these are irregularly spaced compared to silent BESs. rDNA coding regions are also depleted of nucleosomes, relative to the rDNA spacer. In contrast, genes transcribed by Pol II are organized in a more compact, regularly spaced, nucleosomal structure. These observations provide new insight on antigenic variation by showing that chromatin remodeling is an intrinsic feature of BES regulation.
A Photostable Green Fluorescent Protein Variant for Analysis of Protein Localization in
Candida albicans
Authors: Zhang Chengda , Konopka James B.
Published: November 2009

Fusions to the green fluorescent protein (GFP) are an effective way to monitor protein localization. However, altered codon usage in
Candida
species has delayed implementation of new variants. Examination of three new GFP variants in
Candida albicans
showed that one has higher signal intensity and increased resistance to photobleaching.
Control of Flagellar Gene Regulation in
Legionella pneumophila
and Its Relation to Growth Phase
Authors: Albert-Weissenberger Christiane , Sahr Tobias , Sismeiro Odile , Hacker Jörg , Heuner Klaus , Buchrieser Carmen
Published: November 2009

The bacterial pathogen
Legionella pneumophila
responds to environmental changes by differentiation. At least two forms are well described: replicative bacteria are avirulent; in contrast, transmissive bacteria express virulence traits and flagella. Phenotypic analysis, Western blotting, and electron microscopy of mutants of the regulatory genes encoding RpoN, FleQ, FleR, and FliA demonstrated that flagellin expression is strongly repressed and that the mutants are nonflagellated in the transmissive phase. Transcriptome analyses elucidated that RpoN, together with FleQ, enhances transcription of 14 out of 31 flagellar class II genes, which code for the basal body, hook, and regulatory proteins. Unexpectedly, FleQ independent of RpoN enhances the transcription of
fliA
encoding sigma 28. Expression analysis of a
fliA
mutant showed that FliA activates three out of the five remaining flagellar class III genes and the flagellar class IV genes. Surprisingly, FleR does not induce but inhibits expression of at least 14 flagellar class III genes on the transcriptional level. Thus, we propose that flagellar class II genes are controlled by FleQ and RpoN, whereas the transcription of the class III gene
fliA
is controlled in a FleQ-dependent but RpoN-independent manner. However, RpoN and FleR might influence flagellin synthesis on a posttranscriptional level. In contrast to the commonly accepted view that enhancer-binding proteins such as FleQ always interact with RpoN to fullfill their regulatory functions, our results strongly indicate that FleQ regulates gene expression that is RpoN dependent and RpoN independent. Finally, FliA induces expression of flagellar class III and IV genes leading to the complete synthesis of the flagellum.
The Zinc-Responsive Regulator Zur Controls Expression of the Coelibactin Gene Cluster inStreptomyces coelicolor
Authors: Kallifidas Dimitris , Pascoe Ben , Owen Gillian A. , Strain-Damerell Claire M. , Hong Hee-Jeon , Paget Mark S. B.
Published: November 2009

Streptomyces coelicolormutants lacking the zinc-responsive Zur repressor are conditionally defective in sporulation, presumably due to the overexpression of one or more Zur target genes. Gene disruption analyses revealed that deregulation of previously known Zur targets was not responsible for the sporulation phenotype. We used microarrays to identify further Zur targets and discovered that Zur controls a cluster of genes predicted to direct synthesis of an uncharacterized siderophore-related non-ribosomally encoded peptide designated coelibactin. Disruption of a key coelibactin biosynthetic gene suppressed the Zur sporulation phenotype, suggesting that deregulation of coelibactin synthesis inhibits sporulation.
Structure-Function Analysis of IntDOT
Authors: Kim Seyeun , Swalla Brian M. , Gardner Jeffrey F.
Published: November 2009

CTnDOT integrase (IntDOT) is a member of the tyrosine family of site-specific DNA recombinases. IntDOT is unusual in that it catalyzes recombination between nonidentical sequences. Previous mutational analyses centered on mutants with substitutions of conserved residues in the catalytic (CAT) domain or residues predicted by homology modeling to be close to DNA in the core-binding (CB) domain. That work suggested that a conserved active-site residue (Arg I) of the CAT domain is missing and that some residues in the CB domain are involved in catalysis. Here we used a genetic approach and constructed anEscherichia coliindicator strain to screen for random mutations in IntDOT that disrupt integrative recombinationin vivo. Twenty-five IntDOT mutants were isolated and characterized for DNA binding, DNA cleavage, and DNA ligation activities. We found that mutants with substitutions in the amino-terminal (N) domain were catalytically active but defective in forming nucleoprotein complexes, suggesting that they have altered protein-protein interactions or altered interactions with DNA. Replacement of Ala-352 of the CAT domain disrupted DNA cleavage but not DNA ligation, suggesting that Ala-352 may be important for positioning the catalytic tyrosine (Tyr-381) during cleavage. Interestingly, our biochemical data and homology modeling of the CAT domain suggest that Arg-285 is the missing Arg I residue of IntDOT. The predicted position of Arg-285 shows it entering the active site from a position on the polypeptide backbone that is not utilized in other tyrosine recombinases. IntDOT may therefore employ a novel active-site architecture to catalyze recombination.
RecA-Independent DNA Damage Induction of
Mycobacterium tuberculosis ruvC
Despite an Appropriately Located SOS Box
Authors: Dawson Lisa F. , Dillury Joanna , Davis Elaine O.
Published: November 2009

Mycobacterium tuberculosis ruvC
was induced by DNA damage in a Δ
recA
strain despite having an appropriately positioned SOS box to which LexA binds
in vitro
. An inducible transcript start mapped within the SOS box, and transcriptional fusions identified the promoter. Disruption of the SOS box did not prevent induction, indicating that an alternative mechanism plays a significant role in the control of
ruvC
expression.
Triskelion Structure of the Gli521 Protein, Involved in the Gliding Mechanism of
Mycoplasma mobile
Authors: Nonaka Takahiro , Adan-Kubo Jun , Miyata Makoto
Published: November 2009

Mycoplasma mobile
binds to solid surfaces and glides smoothly and continuously by a unique mechanism. A huge protein, Gli521 (521 kDa), is involved in the gliding machinery, and it is localized in the cell neck, the base of the membrane protrusion. This protein is thought to have the role of force transmission. In this study, the Gli521 protein was purified from
M. mobile
cells, and its molecular shape was studied. Gel filtration analysis showed that the isolated Gli521 protein forms mainly a monomer in Tween 80-containing buffer and oligomers in Triton X-100-containing buffer. Rotary shadowing electron microscopy showed that the Gli521 monomer consisted of three parts: an oval, a rod, and a hook. The oval was 15 nm long by 11 nm wide, and the filamentous part composed of the rod and the hook was 106 nm long and 3 nm in diameter. The Gli521 molecules form a trimer, producing a “triskelion” reminiscent of eukaryotic clathrin, through association at the hook end. Image averaging of the central part of the triskelion suggested that there are stable and rigid structures. The binding site of a previously isolated monoclonal antibody on Gli521 images showed that the hook end and oval correspond to the C- and N-terminal regions, respectively. Partial digestion of Gli521 showed that the molecule could be divided into three domains, which we assigned to the oval, rod, and hook of the molecular image. The Gli521 molecule's role in the gliding mechanism is discussed.
Genome-Wide Characterization of the SloR Metalloregulome in
Streptococcus mutans
Authors: O'Rourke Kevin P. , Shaw Jeremy D. , Pesesky Mitchell W. , Cook Brian T. , Roberts Susanne M. , Bond Jeffrey P. , Spatafora Grace A.
Published: November 2009

Streptococcus mutans
is the primary causative agent of human dental caries, a ubiquitous infectious disease for which effective treatment strategies remain elusive. We investigated a 25-kDa SloR metalloregulatory protein in this oral pathogen, along with its target genes that contribute to cariogenesis. Previous studies have demonstrated manganese- and SloR-dependent repression of the
sloABCR
metal ion transport operon in
S. mutans
. In the present study, we demonstrate that
S. mutans
coordinates this repression with that of certain virulence attributes. Specifically, we noted virulence gene repression in a manganese-containing medium when SloR binds to promoter-proximal sequence palindromes on the
S. mutans
chromosome. We applied a genome-wide approach to elucidate the sequences to which SloR binds and to reveal additional “class I” genes that are subject to SloR- and manganese-dependent repression. These analyses identified 204
S. mutans
genes that are preceded by one or more conserved palindromic SloR recognition elements (SREs). We cross-referenced these genes with those that we had identified previously as SloR and/or manganese modulated in microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) experiments. From this analysis, we identified a number of
S. mutans
virulence genes that are subject to transcriptional upregulation by SloR and noted that such “class II”-type regulation is dependent on direct SloR binding to promoter-distal SREs. These observations are consistent with a bifunctional role for the SloR metalloregulator and implicate it as a target for the development of therapies aimed at alleviating
S. mutans
-induced caries formation.
Regulatory Interactions of a Virulence-Associated Serine/Threonine Phosphatase-Kinase Pair in
Bacillus anthracis
Authors: Shakir Salika M. , Bryant Katie M. , Larabee Jason L. , Hamm Elaine E. , Lovchik Julie , Lyons C. Rick , Ballard Jimmy D.
Published: November 2009

In the current study, we examined the regulatory interactions of a serine/threonine phosphatase (BA-Stp1), serine/threonine kinase (BA-Stk1) pair in
Bacillus anthracis
.
B. anthracis
STPK101, a null mutant lacking BA-Stp1 and BA-Stk1, was impaired in its ability to survive within macrophages, and this correlated with an observed reduction in virulence in a mouse model of pulmonary anthrax. Biochemical analyses confirmed that BA-Stp1 is a PP2C phosphatase and dephosphorylates phosphoserine and phosphothreonine residues. Treatment of BA-Stk1 with BA-Stp1 altered BA-Stk1 kinase activity, indicating that the enzymatic function of BA-Stk1 can be influenced by BA-Stp1 dephosphorylation. Using a combination of mass spectrometry and mutagenesis approaches, three phosphorylated residues, T165, S173, and S214, in BA-Stk1 were identified as putative regulatory targets of BA-Stp1. Further analysis found that T165 and S173 were necessary for optimal substrate phosphorylation, while S214 was necessary for complete ATP hydrolysis, autophosphorylation, and substrate phosphorylation. These findings provide insight into a previously undescribed Stp/Stk pair in
B. anthracis
.
Requirements for Germination ofClostridium sordelliiSporesIn Vitro
Authors: Ramirez Norma , Abel-Santos Ernesto
Published: November 2009

Clostridium sordelliiis a spore-forming, obligately anaerobic, Gram-positive bacterium that can cause toxic shock syndrome after gynecological procedures. Although the incidence ofC. sordelliiinfection is low, it is fatal in most cases. Since spore germination is believed to be the first step in the establishment ofBacilliandClostridiainfections, we analyzed the requirements forC. sordelliispore germinationin vitro. Our data showed thatC. sordelliispores require three structurally different amino acids and bicarbonate for maximum germination. Unlike the case forBacillispecies,d-alanine had no effect onC. sordelliispore germination.C. sordelliispores germinated only in a narrow pH range between 5.7 and 6.5. In contrast,C. sordelliispore germination was significantly less sensitive to temperature changes than that of theBacilli. The analysis of the kinetics ofC. sordelliispore germination showed strong allosteric behavior in the binding ofl-phenylalanine andl-alanine but not in that of bicarbonate orl-arginine. By comparing germinant apparent binding affinities to their knownin vivoconcentrations, we postulated a mechanism for differentialC. sordelliispore activation in the female reproductive tract.
Gene Cluster Involved in the Biosynthesis of Griseobactin, a Catechol-Peptide Siderophore of
Streptomyces
sp. ATCC 700974
Authors: Patzer Silke I. , Braun Volkmar
Published: November 2009

The main siderophores produced by streptomycetes are desferrioxamines. Here we show that
Streptomyces
sp. ATCC 700974 and several
Streptomyces griseus
strains, in addition, synthesize a hitherto unknown siderophore with a catechol-peptide structure, named griseobactin. The production is repressed by iron. We sequenced a 26-kb DNA region comprising a siderophore biosynthetic gene cluster encoding proteins similar to DhbABCEFG, which are involved in the biosynthesis of 2,3-dihydroxybenzoate (DHBA) and in the incorporation of DHBA into siderophores via a nonribosomal peptide synthetase. Adjacent to the biosynthesis genes are genes that encode proteins for the secretion, uptake, and degradation of siderophores. To correlate the gene cluster with griseobactin synthesis, the
dhb
genes in ATCC 700974 were disrupted. The resulting mutants no longer synthesized DHBA and griseobactin; production of both was restored by complementation with the
dhb
genes. Heterologous expression of the
dhb
genes or of the entire griseobactin biosynthesis gene cluster in the catechol-negative strain
Streptomyces lividans
TK23 resulted in the synthesis and secretion of DHBA or griseobactin, respectively, suggesting that these genes are sufficient for DHBA and griseobactin biosynthesis. Griseobactin was purified and characterized; its structure is consistent with a cyclic and, to a lesser extent, linear form of the trimeric ester of 2,3-dihydroxybenzoyl-arginyl-threonine complexed with aluminum under iron-limiting conditions. This is the first report identifying the gene cluster for the biosynthesis of DHBA and a catechol siderophore in
Streptomyces
.
Agreement in diagnosing occupational asthma by occupational and respiratory physicians who report to surveillance schemes for work-related ill-health
Authors: Turner Susan , McNamee Roseanne , Roberts Catherine , Bradshaw Lisa , Curran Andrew , Francis Mandy , Fishwick David , Agius Raymond
Published: November 2009

Objectives

To assess diagnostic agreement for occupational asthma, and to identify case and rater characteristics associated with this diagnosis.

Methods

Summaries of possible occupational asthma cases were sent to 104 occupational and respiratory physicians. Raters assigned likelihood scores (0–100%) of occupational asthma based on case histories (phase 1), and on histories plus investigative procedures (phase 2). Interclass correlation coefficients were calculated as statistical measures of reliability for occupational asthma scores. Comparisons between mean scores were assessed for statistical significance using tests based on multilevel models. RRs were calculated to summarise effects of raters' demographics, and of supplying investigative procedures information.

Results

Occupational asthma scores showed limited agreement within each group of (occupational or respiratory) physicians, but scores were not systematically different. The difference between mean overall scores was 2.1% (52.1% occupational physicians; 50.0% respiratory physicians) in phase 1 (95% CI −2.6 to 6.8, p=0.37). In phase 2, mean overall scores were 46.1% (occupational physicians) and 41.5% (respiratory physicians); the difference in mean overall scores was 4.6% (95% CI −3.5 to 12.5, p=0.27). Raters with General Medical Council registration ≥1986 were more likely to give a positive occupational asthma diagnosis. In phase 2, male raters were more likely to label cases as occupational asthma than female raters (RR 4.5, 95% CI 3.3 to 6.0).

Conclusions

The RR of a positive occupational asthma diagnosis was unaffected by clinical speciality. Further work on why physicians consider cases to be occupational asthma will assist better diagnosis and prevention of this disease.
Employee control over working times and risk of cause-specific disability pension: the Finnish Public Sector Study
Authors: Vahtera Jussi , Laine Sari , Virtanen Marianna , Oksanen Tuula , Koskinen Aki , Pentti Jaana , Kivimaki Mika
Published: November 2009

Objective

To examine the association between worktime control and subsequent disability pension among employees.

Methods

Two scores of worktime control (self-assessed and co-worker assessed) were obtained from a survey in 2000–2001 (score range 1–5) among 30 700 public sector employees (78% women) aged 18–64 years. Information on cause-specific disability pension during follow-up was collected from national registers.

Results

During a mean follow-up of 4.4 years, 1178 employees were granted disability pensions (incidence per 1000 person-years: 9.2 in women and 8.7 in men). The most common causes of a disability pension were musculoskeletal disorders (43% of all pensions), mental disorders (25%), tumours (8%) and diseases of the circulatory system (6%) and nervous system (6%). A one unit increase in self-assessed and co-worker assessed worktime control score was associated with a 41–48% decrease in risk of disabling musculoskeletal disorders in men and a 33–35% decrease in women. This association was robust to adjustment for 17 baseline covariates (in men and women combined, adjusted HR 0.76, 95% CI 0.67 to 0.87 and 0.64, 95% CI 0.51 to 0.79 per one unit increase in self-assessed and co-worker assessed worktime control, respectively). Self-assessed, but not co-worker assessed, worktime control was also associated with risk of disability retirement due to mental disorders in women. Disability pensions from other disease categories were not related to worktime control.

Conclusions

In these public sector employees, high worktime control was associated with reduced risk of early retirement caused by musculoskeletal disorders independent of baseline characteristics.
An approach for estimating the health effects of changes over time in air pollution: an illustration using cardio-respiratory hospital admissions in London
Authors: Tonne Cathryn , Beevers Sean , Kelly Frank J , Jarup Lars , Wilkinson Paul , Armstrong Ben
Published: November 2009

Objectives

First, we present a general analytical approach to estimating the association between medium-term changes in air pollution and health across small areas. As a specific illustration, we then applied the approach to data on London residents from a 4-year period to test whether reductions in traffic-related air pollution were associated with reductions in cardio-respiratory hospital admissions.

Methods

A binomial distribution was used to model change in admissions over time in each small area, which was measured as the proportion of admissions in 2003–2004 out of admissions over all study years (2001–2004). Annual average concentrations of nitrogen oxides (NOx) were modelled using an emissions-dispersion model. The association between change in NOxand change in hospital admissions was estimated using logistic regression and an instrumental variable approach.

Results

For some diagnostic groups, suggestive associations between reductions in NOxand reductions in admissions were observed, for example, OR=0.97 (95% CI 0.96 to 0.99) for an IQR decrease in NOx(3 μg/m3) and all respiratory admissions. Accounting for spatial dependence attenuated several of the associations; for respiratory admissions, the OR was 1.00 (95% CI 0.98 to 1.02), leaving only that for bronchiolitis significant (OR=0.91; 95% CI 0.84 to 0.99). In this particular illustration, the instrumental variable approach did not appear to add information.

Conclusions

In this illustration, there was relatively limited power to detect an association between changes in air pollution and hospital admissions over time. However, the analytical approach could deliver more robust estimates of the health effects of changes in air pollution in settings with greater spatial contrast in changes in air pollution over time.
Dermal exposure to polycyclic aromatic hydrocarbons in asphalt workers
Authors: Fustinoni Silvia , Campo Laura , Cirla Piero E , Martinotti Irene , Buratti Marina , Longhi Omar , Foà Vito , Bertazzi PierAlberto
Published: November 2009

Objectives

To assess dermal exposure to 16 polycyclic aromatic hydrocarbons (PAHs) in asphalt workers by applying polypropylene pads to six body sites (neck, shoulder, upper arm, wrist, groin, ankle), to identify the compounds and exposure sites most representative, and to integrate dermal exposure results with environmental and biological data.

Methods

Twenty-four asphalt workers were recruited. Dermal exposure was assessed during a single work shift. Sixteen PAHs (from naphthalene to indeno[1,2,3-cd]pyrene) were quantified via gas chromatography-mass spectrometry. Airborne exposure, urinary PAHs and monohydroxy metabolites were also investigated.

Results

Phenanthrene (PHE), present in all samples, was the most abundant compound (median 0.805–1.825 ng/cm2). Benzo[a]pyrene (BaP) was present in 75% of the samples (0.046–0.101 ng/cm2). Wrist had the highest contamination, with median PHE, pyrene (PYR), and BaP concentrations of 1.825, 0.527, and 0.063 ng/cm2. PHE and PYR on wrist correlated with almost all 3- to 4-ring PAHs (0.405≤r≤0.856), but not with BaP; BaP correlated with almost all 4- to 6- ring PAHs (0.584≤r≤0.633). Significant correlations were observed between PHE level, airborne exposure, and the corresponding urinary PHE and monohydroxy metabolites. For PYR, significant correlations existed only between urinary PYR and monohydroxy metabolites. Multiple linear regression analysis revealed that 42% of the end-of-shift monohydroxy metabolites were the result of airborne exposure, dermal exposure, and baseline levels of biomarkers.

Conclusions

Dermal exposure to PAHs was in the low ng/cm2range. PHE or PYR and BaP were the most representative compounds and the wrist was the best location to perform dermal exposure assessments. Both dermal and airborne exposure contributed to the total body burden of PAHs, though the relative contribution was analyte-dependent.
Selecting an appropriate biomonitoring strategy to evaluate dermal exposure to opioid narcotic analgesics in pharmaceutical production workers
Authors: Van Nimmen N F J , Poels K L C , Severi M J , Godderis L , Veulemans H A F
Published: November 2009

Objectives

In a follow-up study of previous research, in which exposure pathways for opioid narcotic analgesics were identified in pharmaceutical workers involved in drug synthesis, the current research focused on the selection of an appropriate biomonitoring strategy.

Methods

Six opioid narcotic production workers were intensively monitored during a (1 week) fentanyl production campaign. A systematic sampling scheme was followed that provided information about hand contamination and biomarker levels at multiple time points.

Results

Linear mixed-effects models, incorporating half-shift and end-of-shift hand contamination levels, showed a positive and significant correlation with fentanyl urinary excretion occurring at many of the 4 h time lags investigated (4–28 h). Optimum model characteristics, including both minimal between- and within-worker variability, were obtained at lag times of 24 h and 20 h, respectively, advocating a pre-shift urine sampling strategy on the following day. In addition, for these lag times the portion of the variability explained by the model was maximal. Furthermore, using a distributed lag model, it was demonstrated that urinary fentanyl levels were positively correlated with hand contamination levels measured at the preceding four 8 h time lags (8–32 h), although statistical significance was only shown for a lag time of 24 h.

Conclusion

Fentanyl levels in pre-shift urine samples reflect dermal exposure to the compound during the previous day. Thus, in the specific working environment investigated, a biological monitoring protocol evaluating pre-shift urinary fentanyl levels could provide an adequate risk estimate in individual workers.