bluuebunny/crossref_metadata_2025 huggingface dataset:
Alternative splicing expands the repertoire of dominant JAZ repressors of jasmonate signaling
Authors: Chung Hoo Sun , Cooke Thomas F. , DePew Cody L. , Patel Lalita C. , Ogawa Narihito , Kobayashi Yuichi , Howe Gregg A.
Published: May 2010

Jasmonates (JAs) are fatty acid‐derived signaling compounds that control diverse aspects of plant growth, development and immunity. The F‐box protein COI1 functions both as a receptor for jasmonoyl‐l‐isoleucine (JA‐Ile) and as the component of an E3‐ubiquitin ligase complex (SCFCOI1) that targets JAZ transcriptional regulators for degradation. A key feature of JAZ proteins is the C‐terminal Jas motif that mediates the JA‐Ile‐dependent interaction with COI1. Here, we show that most JAZ genes from evolutionarily diverse plants contain a conserved intron that splits the Jas motif into 20 N‐terminal and seven C‐terminal (X5PY) amino acid submotifs. In most members of the Arabidopsis JAZ family, alternative splicing events involving retention of this intron generate proteins that are truncated before the X5PY sequence. In vitro pull‐down and yeast two‐hybrid assays indicate that these splice variants have reduced capacity to form stable complexes with COI1 in the presence of the bioactive stereoisomer of the hormone (3R,7S)‐JA‐Ile. cDNA overexpression studies showed that some, but not all, truncated splice variants are dominant repressors of JA signaling. We also show that strong constitutive expression of an intron‐containing JAZ10 genomic clone is sufficient to repress JA responses. These findings provide evidence for functional differences between JAZ isoforms, and establish a direct link between the alternative splicing of JAZ pre‐mRNA and the dominant repression of JA signal output. We propose that production of dominant JAZ repressors by alternative splicing reduces the negative consequences associated with inappropriate or hyperactivation of the JA response pathway.
Measuring the turnover rates of Arabidopsis proteins using deuterium oxide: an auxin signaling case study
Authors: Yang Xiao‐Yuan , Chen Wen‐Ping , Rendahl Aaron K. , Hegeman Adrian D. , Gray William M. , Cohen Jerry D.
Published: May 2010

Rapid environmental responses in plants rely on endogenous signaling mechanisms, which in many cases are mediated by changes in protein turnover rates. It is therefore necessary to develop methods for measuring protein dynamics that monitor large sets of plant proteins to begin to apply a systems biology approach to the study of plant behavior. The use of stable isotope labeling strategies that are adaptable to proteomic methods is particularly attractive for this purpose. Here, we explore one example of such methods that is particularly suitable for plants at the seedling stage, where measurement of amino acid and protein turnover rates is accomplished using a heavy water labeling strategy. The method is backed by microarray evaluation to define its feasibility for specific experimental approaches, and the CULLIN‐ASSOCIATED AND NEDDYLATION DISSOCIATED 1 (CAND1) and TRANSPORT INHIBITOR RESPONSE 1 (TIR1) proteins are used to illustrate the potential utility in understanding hormonal signaling regulation. These studies provide insight not only into the potential utility of the method, but also address possible areas of concern regarding the use of heavy water labeling during plant growth. These considerations suggest a prescription for specific experimental designs that minimize interference resulting from the induction of treatment‐specific gene expression in the results obtained.
Clinical significance of occult cerebrospinal fluid involvement assessed by flow cytometry in non‐Hodgkin’s lymphoma patients at high risk of central nervous system disease in the rituximab era
Authors: Sancho Juan‐Manuel , Orfao Alberto , Quijano Sandra , García Olga , Panizo Carlos , Pérez‐Ceballos Elena , Deben Guillermo , Salar Antonio , González‐Barca Eva , Alonso Natalia , García‐Vela Jose‐Antonio , Capote Javier , Peñalver Francisco‐Javier , Provencio Mariano , Arias Jesús , Plaza Josefa , Caballero Dolores , Morado Marta , Feliu Evarist , Ribera Josep‐Maria ,   for the Spanish Group for the Study of CNS Disease in NHL
Published: May 2010

Background and aim:  Flow cytometry (FCM) analysis of cerebrospinal fluid (CSF) is more sensitive than conventional cytology (CC) for diagnosis of lymphomatous meningeosis, but the clinical significance of occult central nervous system (CNS) disease (positive FCM with negative CC) remains unknown.

Patients and methods:  CSF samples from 105 patients with newly diagnosed aggressive lymphomas at high risk of CNS involvement were prospectively studied by both CC and FCM, and results were correlated with cumulative incidence of CNS relapse and overall survival (OS). Patients were divided into three groups: 1) patients without CNS involvement (CC−/FCM−; n = 83); 2) individuals with occult CNS disease (FCM+/CC−; n = 15); and 3) cases with CNS disease (CC+/FCM+; n = 7).

Results:  Six cases showed CNS relapse or progression: two in Group 1 (2.4%), two in Group 2 (13%) and two in Group 3 (28.5%) (Group 2 vs. 1, P = 0.04; Group 3 vs. 1, P < 0.001). Patients from Groups 2 (P = 0.05) and 3 (P < 0.001) also showed a higher cumulative incidence of CNS relapse than those from Group 1. Significant differences were observed in OS between FCM−/CC− and FCM+/CC+ cases (P = 0.02), while patients with occult CNS disease (FCM+/CC−) displayed intermediate OS rates, although differences did not reach statistical significance.

Conclusions:  The presence of occult CNS involvement at diagnosis in patients with NHL at high risk of CNS disease is associated with a higher probability of CNS relapse.
Maternal serum hepcidin is low at term and independent of cord blood iron status
Authors: Rehu Mari , Punnonen Kari , Ostland Vaughn , Heinonen Seppo , Westerman Mark , Pulkki Kari , Sankilampi Ulla
Published: May 2010

Objectives:  Hepcidin is the key regulator of iron homeostasis. The aims of this study were to determine serum hepcidin concentrations and reference ranges in pregnant women and cord blood of newborns at term and to evaluate the associations between hepcidin concentrations and iron status parameters.

Methods:  A total of 191 pregnant women–newborn pairs were studied in Kuopio University Hospital, Finland. The measured parameters were serum hepcidin, ferritin, transferrin receptor, transferrin saturation, red cell indices, and erythropoietin.

Results:  The hepcidin concentration in pregnant women was significantly lower than in cord blood at term [geometric mean concentration (GMC) (95% confidence intervals) in pregnant women 10.7 ng/mL (8.5–13.4 ng/mL) vs. GMC of cord blood hepcidin 69.3 ng/mL (55.3–86.8 ng/mL), P < 0.001, adjusted analysis of variance]. Hepcidin was undetectable in 12% of mothers. Hepcidin concentration in pregnant women was the lowest in those who had the lowest iron status. However, maternal hepcidin concentration was not associated with cord blood hepcidin or iron status markers. Hepcidin concentration in cord blood was associated with cord blood iron status, but not with maternal iron status.

Conclusions:  At term pregnancy, hepcidin concentrations are very low, allowing maximal availability of iron for the fetus. Maternal and cord blood hepcidin levels were independently associated with either maternal or cord blood iron status.
Stimulation of duodenal HCO3−secretion by hydrogen sulphide in rats: relation to prostaglandins, nitric oxide and sensory neurones
Authors: Ise F. , Takasuka H. , Hayashi S. , Takahashi K. , Koyama M. , Aihara E. , Takeuchi K.
Published: May 2010

Aim: We examined the effect of H2S on duodenal HCO3−secretion in rats and investigated the mechanism involved in this response.

Methods: Animals were fasted for 18 h and anaesthetized with urethane. A duodenal loop was perfused with saline, and HCO3−secretion was measured at pH 7.0 using a pH stat‐method. The loop was perfused at a rate of 0.2 mL min−1with NaHS (H2S donor: 0.1–1 mm) for 5 min or 10 mmHCl for 10 min. Indomethacin orl‐NAME [nitric oxide (NO) synthase inhibitor) was given s.c. 30 min or 3 h, respectively, before NaHS or acidification, while glibenclamide (KATPchannel blocker) or propargylglycine (cystathionine‐g‐lyase inhibitor) was given i.p. 30 min before.

Results: Mucosal perfusion with NaHS dose dependently increased the HCO3−secretion, and this effect was significantly attenuated by indomethacin andl‐NAME as well as by sensory deafferentation, but not by glibenclamide. Mucosal prostaglandin E2(PGE2) production and luminal release of NO were both increased by NaHS perfusion. Mucosal acidification stimulated HCO3−secretion concomitant with an increase in PGE2and NO production, and these responses were mitigated by propargylglycine. The duodenal damage induced by acid (100 mmHCl for 4 h) was aggravated by pre‐treatment with propargylglycine.

Conclusion: These results suggest that H2S increases HCO3−secretion in the rat duodenum, and that this action is partly mediated by PG and NO as well as by capsaicin‐sensitive afferent neurones. It is assumed that endogenous H2S is involved in the regulatory mechanism of acid‐induced HCO3−secretion and mucosal protection in the duodenum.
Dynamic characteristics of heart rate control by the autonomic nervous system in rats
Authors: Mizuno Masaki , Kawada Toru , Kamiya Atsunori , Miyamoto Tadayoshi , Shimizu Shuji , Shishido Toshiaki , Smith Scott A. , Sugimachi Masaru
Published: May 2010

We estimated the transfer function of autonomic heart rate (HR) control by using random binary sympathetic or vagal nerve stimulation in anaesthetized rats. The transfer function from sympathetic stimulation to HR response approximated a second‐order, low‐pass filter with a lag time (gain, 4.29 ± 1.55 beats min−1 Hz−1; natural frequency, 0.07 ± 0.03 Hz; damping coefficient, 1.96 ± 0.64; and lag time, 0.73 ± 0.12 s). The transfer function from vagal stimulation to HR response approximated a first‐order, low‐pass filter with a lag time (gain, 8.84 ± 4.51 beats min−1 Hz−1; corner frequency, 0.12 ± 0.06 Hz; and lag time, 0.12 ± 0.08 s). These results suggest that the dynamic characteristics of HR control by the autonomic nervous system in rats are similar to those of larger mammals.
Beneficial Effect of
Verminephrobacter
Nephridial Symbionts on the Fitness of the Earthworm
Aporrectodea tuberculata
Authors: Lund Marie B. , Holmstrup Martin , Lomstein Bente A. , Damgaard Christian , Schramm Andreas
Published: May 2010

Almost all lumbricid earthworms (Oligochaeta: Lumbricidae) harbor species-specific
Verminephrobacter
(
Betaproteobacteria
) symbionts in their nephridia (excretory organs). The function of the symbiosis, and whether the symbionts have a beneficial effect on their earthworm host, is unknown; however, the symbionts have been hypothesized to enhance nitrogen retention in earthworms. The effect of
Verminephrobacter
on the life history traits of the earthworm
Aporrectodea tuberculata
(Eisen) was investigated by comparing the growth, development, and fecundity of worms with and without symbionts given high (cow dung)- and low (straw)-nutrient diets. There were no differences in worm growth or the number of cocoons produced by symbiotic and aposymbiotic worms. Worms with
Verminephrobacter
symbionts reached sexual maturity earlier and had higher cocoon hatching success than worms cured of their symbionts when grown on the low-nutrient diet. Thus,
Verminephrobacter
nephridial symbionts do have a beneficial effect on their earthworm host. Cocoons with and without symbionts did not significantly differ in total organic carbon, total nitrogen, or total hydrolyzable amino acid content, which strongly questions the hypothesized role of the symbionts in nitrogen recycling for the host.
Quantitative Real-Time PCR Assays for Sensitive Detection of Canada Goose-Specific Fecal Pollution in Water Sources
Authors: Fremaux B. , Boa T. , Yost C. K.
Published: May 2010

Canada geese (
Branta canadensis
) are prevalent in North America and may contribute to fecal pollution of water systems where they congregate. This work provides two novel real-time PCR assays (CGOF1-
Bac
and CGOF2-
Bac
) allowing for the specific and sensitive detection of
Bacteroides
16S rRNA gene markers present within Canada goose feces.
Enrichment and Physiological Characterization of a Novel
Nitrospira
-Like Bacterium Obtained from a Marine Sponge
Authors: Off Sandra , Alawi Mashal , Spieck Eva
Published: May 2010

Members of the nitrite-oxidizing genus
Nitrospira
are most likely responsible for the second step of nitrification, the conversion of nitrite (NO
2
−
) to nitrate (NO
3
−
), within various sponges. We succeeded in obtaining an enrichment culture of
Nitrospira
derived from the mesohyl of the marine sponge
Aplysina aerophoba
using a traditional cultivation approach. Electron microscopy gave first evidence of the shape and ultrastructure of this novel marine
Nitrospira
-like bacterium (culture Aa01). We characterized these bacteria physiologically with regard to optimal incubation conditions, especially the temperature and substrate range in comparison to other
Nitrospira
cultures. Best growth was obtained at temperatures between 28°C and 30°C in mineral medium with 70% North Sea water and a substrate concentration of 0.5 mM nitrite under microaerophilic conditions. The
Nitrospira
culture Aa01 is very sensitive against nitrite, because concentrations higher than 1.5 mM resulted in a complete inhibition of growth. Sequence analyses of the 16S rRNA gene revealed that the novel
Nitrospira
-like bacterium is separated from the sponge-specific subcluster and falls together with an environmental clone from Mediterranean sediments (98.6% similarity). The next taxonomically described species
Nitrospira marina
is only distantly related, with 94.6% sequence similarity, and therefore the culture Aa01 represents a novel species of nitrite-oxidizing bacteria.
Coupled Arsenotrophy in a Hot Spring Photosynthetic Biofilm at Mono Lake, California
Authors: Hoeft Shelley E. , Kulp Thomas R. , Han Sukkyun , Lanoil Brian , Oremland Ronald S.
Published: May 2010

Red-pigmented biofilms grow on rock and cobble surfaces present in anoxic hot springs located on Paoha Island in Mono Lake. The bacterial community was dominated (∼ 85% of 16S rRNA gene clones) by sequences from the photosynthetic
Ectothiorhodospira
genus. Scraped biofilm materials incubated under anoxic conditions rapidly oxidized As(III) to As(V) in the light via anoxygenic photosynthesis but could also readily reduce As(V) to As(III) in the dark at comparable rates. Back-labeling experiments with
73
As(V) demonstrated that reduction to
73
As(III) also occurred in the light, thereby illustrating the cooccurrence of these two anaerobic processes as an example of closely coupled arsenotrophy. Oxic biofilms also oxidized As(III) to As(V). Biofilms incubated with [
14
C]acetate oxidized the radiolabel to
14
CO
2
in the light but not the dark, indicating a capacity for photoheterotrophy but not chemoheterotrophy. Anoxic, dark-incubated samples demonstrated As(V) reduction linked to additions of hydrogen or sulfide but not acetate. Chemoautotrophy linked to As(V) as measured by dark fixation of [
14
C]bicarbonate into cell material was stimulated by either H
2
or HS
−
. Functional genes for the arsenate respiratory reductase (
arrA
) and arsenic resistance (
arsB
) were detected in sequenced amplicons of extracted DNA, with about half of the
arrA
sequences closely related (∼98% translated amino acid identity) to those from the family
Ectothiorhodospiraceae
. Surprisingly, no authentic PCR products for arsenite oxidase (
aoxB
) were obtained, despite observing aerobic arsenite oxidation activity. Collectively, these results demonstrate close linkages of these arsenic redox processes occurring within these biofilms.
Antibiotic Resistance Markers for Genetic Manipulations of
Leptospira
spp
Authors: Poggi Dante , Oliveira de Giuseppe Priscila , Picardeau Mathieu
Published: May 2010

We measured the frequency of appearance of spontaneous mutants resistant to gentamicin, kanamycin, streptomycin, and spectinomycin in saprophytic and pathogenic
Leptospira
strains. The mutations responsible for the spontaneous resistance to streptomycin and spectinomycin were identified in the
rpsL
and
rrs
genes, respectively. We also generated a gentamicin resistance cassette that allows the use of a third selectable marker in leptospires. These results may facilitate further advances in gene transfer systems in
Leptospira
spp.
Spatiotemporal Variations in Microcystin Concentrations and in the Proportions of Microcystin-Producing Cells in Several
Microcystis aeruginosa
Populations
Authors: Sabart M. , Pobel D. , Briand E. , Combourieu B. , Salençon M. J. , Humbert J. F. , Latour D.
Published: May 2010

With the aim of explaining the variations in microcystin (MC) concentrations during cyanobacterial blooms, we studied several
Microcystis aeruginosa
populations blooming in different freshwater ecosystems located in the same geographical area. As assessed by real-time PCR, it appeared that the potentially MC-producing cells (
mcyB
+
) were predominant (70 to 100%) in all of these
M. aeruginosa
populations, with the exception of one population in which non-MC-producing cells always dominated. Apart from the population in the Grangent Reservoir, we found that the proportions of potentially MC-producing and non-MC-producing cells varied little over time, which was consistent with the fact that according to a previous study of the same populations, the intergenic transcribed spacer (ITS) genotype composition did not change (38). In the Grangent Reservoir, the MC-RR variant was the dominant microcystin variant throughout the bloom season, despite changes in the ITS composition and in the proportions of
mcyB
+
cells. Finally, the variations in total MC concentrations (0.3 to 15 μg liter
−1
) and in the MC cellular quotas (0.01 to 3.4 pg cell
−1
) were high both between and within sites, and no correlation was found between the MC concentrations and the proportion of
mcyB
+
cells. All of these findings demonstrate that very different results can be found for the proportions of potentially MC-producing and non-MC-producing cells and MC concentrations, even in
M. aeruginosa
populations living in more or less connected ecosystems, demonstrating the importance of the effect of very local environmental conditions on these parameters and also the difficulty of predicting the potential toxicity of
Microcystis
blooms.
Diversity, Abundance, and Spatial Distribution of Sediment Ammonia-OxidizingBetaproteobacteriain Response to Environmental Gradients and Coastal Eutrophication in Jiaozhou Bay, China
Authors: Dang Hongyue , Li Jing , Chen Ruipeng , Wang Lin , Guo Lizhong , Zhang Zhinan , Klotz Martin G.
Published: May 2010

Ongoing anthropogenic eutrophication of Jiaozhou Bay offers an opportunity to study the influence of human activity on bacterial communities that drive biogeochemical cycling. Nitrification in coastal waters appears to be a sensitive indicator of environmental change, suggesting that function and structure of the microbial nitrifying community may be associated closely with environmental conditions. In the current study, theamoAgene was used to unravel the relationship between sediment aerobic obligate ammonia-oxidizingBetaproteobacteria(Beta-AOB) and their environment in Jiaozhou Bay. Protein sequences deduced fromamoAgene sequences grouped within four distinct clusters in theNitrosomonaslineage, including a putative new cluster. In addition, AmoA sequences belonging to three newly defined clusters in theNitrosospiralineage were also identified. Multivariate statistical analyses indicated that the studied Beta-AOB community structures correlated with environmental parameters, of which nitrite-N and sediment sand content had significant impact on the composition, structure, and distribution of the Beta-AOB community. BothamoAclone library and quantitative PCR (qPCR) analyses indicated that continental input from the nearby wastewater treatment plants and polluted rivers may have significant impact on the composition and abundance of the sediment Beta-AOB assemblages in Jiaozhou Bay. Our work is the first report of a direct link between a sedimentological parameter and the composition and distribution of the sediment Beta-AOB and indicates the potential for using the Beta-AOB community composition in general and individual isolates or environmental clones in theNitrosomonas oligotrophalineage in particular as bioindicators and biotracers of pollution or freshwater or wastewater input in coastal environments.
Novel Assay To Assess Permissiveness of a Soil Microbial Community toward Receipt of Mobile Genetic Elements
Authors: Musovic Sanin , Dechesne Arnaud , Sørensen Jan , Smets Barth F.
Published: May 2010

There is a wealth of evidence indicating that mobile genetic elements can spread in natural microbial communities. However, little is known regarding the fraction of the community that actually engages in this behavior. Here we report on a new approach to quantify the fraction of a bacterial community that is able to receive and maintain an exogenous conjugal plasmid termed community permissiveness. Conjugal transfer of a broad-host-range plasmid labeled with a zygotically inducible green fluorescent protein (RP4::
gfp
) from a donor strain (
Pseudomonas putida
) to a soil bacterial suspension was examined. The mixture of cells was incubated on membrane filters supported by different solid media. Plasmid transfer was scored by
in situ
visualization of green fluorescent transconjugant microcolonies, and host range was determined by traditional plating or microcolony isolation by using a micromanipulator. Among the conditions tested, the highest plasmid transfer incidence (approximately 1 transfer per 10
4
soil bacteria) was measured after 48 h of incubation on either a 10% soil extract or a 10-fold diluted R2A medium. Stereomicroscopy combined with image analysis allowed easy examination and enumeration of green fluorescent microcolonies. In all experiments, however, stereomicroscopy consistently underestimated the number of conjugation events (approximately 10-fold) in comparison to confocal laser scanning microscopy. The plasmid host range was broad and included bacteria belonging to the
Alphaproteobacteria
,
Betaproteobacteria
, and
Gammaproteobacteria
classes of proteobacteria. The isolation of transconjugant microcolonies by micromanipulation greatly extended the estimated plasmid host range among soil bacteria. The new approach can be applied to examine the permissiveness of various communities toward receipt of different mobile elements.
Improvement of Phosphate Solubilization and
Medicago
Plant Yield by an Indole-3-Acetic Acid-Overproducing Strain of
Sinorhizobium meliloti
Authors: Bianco Carmen , Defez Roberto
Published: May 2010

Nitrogen (N) and phosphorus (P) are the most limiting factors for plant growth. Some microorganisms improve the uptake and availability of N and P, minimizing chemical fertilizer dependence. It has been published that the RD64 strain, a
Sinorhizobium meliloti
1021 strain engineered to overproduce indole-3-acetic acid (IAA), showed improved nitrogen fixation ability compared to the wild-type 1021 strain. Here, we present data showing that RD64 is also highly effective in mobilizing P from insoluble sources, such as phosphate rock (PR). Under P-limiting conditions, the higher level of P-mobilizing activity of RD64 than of the 1021 wild-type strain is connected with the upregulation of genes coding for the high-affinity P transport system, the induction of acid phosphatase activity, and the increased secretion into the growth medium of malic, succinic, and fumaric acids.
Medicago truncatula
plants nodulated by RD64 (
Mt
-RD64), when grown under P-deficient conditions, released larger amounts of another P-solubilizing organic acid, 2-hydroxyglutaric acid, than plants nodulated by the wild-type strain (
Mt
-1021). It has already been shown that
Mt
-RD64 plants exhibited higher levels of dry-weight production than
Mt
-1021 plants. Here, we also report that P-starved
Mt
-RD64 plants show significant increases in both shoot and root fresh weights when compared to P-starved
Mt
-1021 plants. We discuss how, in a
Rhizobium
-legume model system, a balanced interplay of different factors linked to bacterial IAA overproduction rather than IAA production
per se
stimulates plant growth under stressful environmental conditions and, in particular, under P starvation.
Mosaic-Like Sequences Containing Transposon, Phage, and Plasmid Elements among
Listeria monocytogenes
Plasmids
Authors: Canchaya Carlos , Giubellini Vanessa , Ventura Marco , de los Reyes-Gavilán Clara G. , Margolles Abelardo
Published: May 2010

Sequencing of plasmid pLM33 from the food isolate
Listeria monocytogenes
Lm1 revealed a molecule of 32,307 bp with a G+C content of 36.2%. The plasmid displays a mosaic pattern of identities common to several closely related
L. monocytogenes
plasmids isolated from food and clinical sources.
Multigeneration Cross Contamination of Mail with
Bacillus
Species Spores by Tumbling
Authors: Edmonds Jason , Clark Paul , Williams Leslie , Lindquist H. D. Alan , Martinez Kenneth , Gardner Warren , Shadomy Sean , Hornsby-Myers Jennifer
Published: May 2010

In 2001, envelopes loaded with
Bacillus anthracis
spores were mailed to Senators Daschle and Leahy as well as to the New York Post and NBC News buildings. Additional letters may have been mailed to other news agencies because there was confirmed anthrax infection of employees at these locations. These events heightened the awareness of the lack of understanding of the mechanism(s) by which objects contaminated with a biological agent might spread disease. This understanding is crucial for the estimation of the potential for exposure to ensure the appropriate response in the event of future attacks. In this study, equipment to simulate interactions between envelopes and procedures to analyze the spread of spores from a “payload” envelope (i.e., loaded internally with a powdered spore preparation) onto neighboring envelopes were developed. Another process to determine whether an aerosol could be generated by opening contaminated envelopes was developed. Subsequent generations of contaminated envelopes originating from a single payload envelope showed a consistent two-log decrease in the number of spores transferred from one generation to the next. Opening a tertiary contaminated envelope resulted in an aerosol containing 10
3
B. anthracis
spores. We developed a procedure for sampling contaminated letters by a nondestructive method aimed at providing information useful for consequence management while preserving the integrity of objects contaminated during the incident and preserving evidence for law enforcement agencies.
Influence of Forest Trees on the Distribution of Mineral Weathering-Associated Bacterial Communities of the
Scleroderma citrinum
Mycorrhizosphere
Authors: Calvaruso Christophe , Turpault Marie-Pierre , Leclerc Elisabeth , Ranger Jacques , Garbaye Jean , Uroz Stéphane , Frey-Klett Pascale
Published: May 2010

In acidic forest soils, availability of inorganic nutrients is a tree-growth-limiting factor. A hypothesis to explain sustainable forest development proposes that tree roots select soil microbes involved in central biogeochemical processes, such as mineral weathering, that may contribute to nutrient mobilization and tree nutrition. Here we showed, by combining soil analyses with cultivation-dependent analyses of the culturable bacterial communities associated with the widespread mycorrhizal fungus
Scleroderma citrinum
, a significant enrichment of bacterial isolates with efficient mineral weathering potentials around the oak and beech mycorrhizal roots compared to bulk soil. Such a difference did not exist in the rhizosphere of Norway spruce. The mineral weathering ability of the bacterial isolates was assessed using a microplaque assay that measures the pH and the amount of iron released from biotite. Using this microplate assay, we demonstrated that the bacterial isolates harboring the most efficient mineral weathering potential belonged to the
Burkholderia
genus. Notably, previous work revealed that oak and beech harbored very similar pHs in the 5- to 10-cm horizon in both rhizosphere and bulk soil environments. In the spruce rhizosphere, in contrast, the pH was significantly lower than that in bulk soil. Because the production of protons is one of the main mechanisms responsible for mineral weathering, our results suggest that certain tree species have developed indirect strategies for mineral weathering in nutrient-poor soils, which lie in the selection of bacterial communities with efficient mineral weathering potentials.
Unsuitability of Quantitative
Bacteroidales
16S rRNA Gene Assays for Discerning Fecal Contamination of Drinking Water
Authors: van der Wielen Paul W. J. J. , Medema Gertjan
Published: May 2010

Bacteroidales
species were detected in (tap) water samples from treatment plants with three different PCR assays. 16S rRNA gene sequence analysis indicated that the sequences had an environmental rather than fecal origin. We conclude that assays for
Bacteroidales
16S rRNA genes are not specific enough to discern fecal contamination of drinking water in the Netherlands.
Sequence Diversity of Genes Encoding Botulinum Neurotoxin Type F
Authors: Raphael Brian H. , Choudoir Mallory J. , Lúquez Carolina , Fernández Rafael , Maslanka Susan E.
Published: May 2010

Botulism due to type F botulinum neurotoxin (BoNT/F) is rare (<1% of cases), and only a limited number of clostridial strains producing this toxin type have been isolated. As a result, analysis of the diversity of genes encoding BoNT/F has been challenging. In this study, the entirebont/Fnucleotide sequences were determined from 33 type F botulinum toxin-producing clostridial strains isolated from environmental sources and botulism outbreak investigations. We examined proteolytic and nonproteolyticClostridium botulinumtype F strains, bivalent strains, including Bf and Af, andClostridium baratiitype F strains. Phylogenetic analysis revealed that thebont/Fgenes examined formed 7 subtypes (F1 to F7) and that the nucleotide sequence identities of these subtypes differed by up to 25%. The genes from proteolytic (group I)C. botulinumstrains formed subtypes F1 through F5, while the genes from nonproteolytic (group II)C. botulinumstrains formed subtype F6. Subtype F7 was composed exclusively ofbont/Fgenes fromC. baratiistrains. The region of thebont/F5gene encoding the neurotoxin light chain was found to be highly divergent compared to the other subtypes. Although thebont/F5nucleotide sequences were found to be identical in strains harboring this gene, the gene located directly upstream (ntnh/F) demonstrated sequence variation among representative strains of this subtype. These results demonstrate that extensive nucleotide diversity exists among genes encoding type F neurotoxins from strains with different phylogenetic backgrounds and from various geographical sources.
The Virulence Regulator PrfA Promotes Biofilm Formation byListeria monocytogenes
Authors: Lemon Katherine P. , Freitag Nancy E. , Kolter Roberto
Published: May 2010

Listeria monocytogenesis a food-borne facultative intracellular pathogen. It is widespread in the environment and has several distinct life-styles. The key transcriptional activator PrfA positively regulatesL. monocytogenesvirulence genes to mediate the transition from extracellular, flagellum-propelled cell to intracellular pathogen. Here we report the first evidence that PrfA also has a significant positive impact on extracellular biofilm formation. Mutants lackingprfAwere defective in surface-adhered biofilm formation. The ΔprfAmutant exhibited wild-type flagellar motility, and its biofilm defect occurred after initial surface adhesion. We also observed that mutations that led to the constitutive expression of PrfA-dependent virulence genes had a minimal impact on biofilm formation. Furthermore, biofilm development was enhanced in a mutant encoding a PrfA protein variant unable to fully transition from the extracellular form to the virulent, intracellular activity conformation. These results indicate that PrfA positively regulates biofilm formation and suggest that PrfA has a global role in modulating the life-style ofL. monocytogenes. The requirement of PrfA for optimal biofilm formation may provide selective pressure to maintain this critical virulence regulator whenL. monocytogenesis outside host cells in the environment.
Identification of
trans
- and
cis
-Control Elements Involved in Regulation of the Carbon Monoxide Dehydrogenase Genes in
Mycobacterium
sp. Strain JC1 DSM 3803
Authors: Oh Jeong-Il , Park Su-Jeong , Shin Sun-Joo , Ko In-Jeong , Han Seung J. , Park Sae W. , Song Taeksun , Kim Young M.
Published: May 2010

The
cutR
gene was identified 314 bp upstream of the divergently oriented
cutB1C1A1
operon encoding carbon monoxide (CO) dehydrogenase in
Mycobacterium
sp. strain JC1. Its deduced product was composed of 320 amino acid residues with a calculated molecular mass of 34.1 kDa and exhibits a basal sequence similarity to the regulatory proteins belonging to the LysR family. Using a
cutR
deletion mutant, it was demonstrated that CutR is required for the efficient utilization of CO by
Mycobacterium
sp. strain JC1 growing with CO as the sole source of carbon and energy. CutR served as a transcriptional activator for expression of the duplicated
cutBCA
operons (
cutB1C1A1
and
cutB2C2A2
) and was involved in the induction of the
cutBCA
operons by CO. The
cutBCA
operons were also subjected to catabolite repression. An inverted repeat sequence (TGTGA-N
6
-TCACA) with a perfect match with the binding motif of cyclic AMP receptor protein was identified immediately upstream of and overlapping with the translational start codons of
cutB1
and
cutB2
. This palindrome sequence was shown to be involved in catabolite repression of the
cutBCA
operons. The transcription start point of
cutR
was determined to be the nucleotide G located 36 bp upstream of the start codon of
cutR
. Expression of
cutR
was higher in
Mycobacterium
sp. strain JC1 grown with glucose than that grown with CO.
Role of MrkJ, a Phosphodiesterase, in Type 3 Fimbrial Expression and Biofilm Formation in
Klebsiella pneumoniae
Authors: Johnson Jeremiah G. , Clegg Steven
Published: May 2010

Klebsiella pneumoniae
is an opportunistic pathogen that has been shown to adhere to human extracellular matrices using the type 3 fimbriae. Introduction of plasmids carrying genes known to alter intracellular cyclic-di-GMP pools in
Vibrio parahaemolyticus
revealed that these genes also altered type 3 fimbrial surface expression in
K. pneumoniae
. Immediately adjacent to the type 3 fimbrial gene cluster is a gene,
mrkJ
, that is related to a family of bacterial genes encoding phosphodiesterases. We identify here a role for MrkJ, a functional phosphodiesterase exhibiting homology to EAL domain-containing proteins, in controlling type 3 fimbria production and biofilm formation in
K. pneumoniae
. Deletion of
mrkJ
resulted in an increase in type 3 fimbria production and biofilm formation as a result of the accumulation of intracellular cyclic-di-GMP. This gene was shown to encode a functional phosphodiesterase via restoration of motility in a
V. parahaemolyticus
strain previously shown to accumulate cyclic-di-GMP and
in vitro
using phosphodiesterase activity assays. The effect of the
mrkJ
mutation on type 3 fimbrial expression was shown to be at the level of
mrkA
gene transcription by using quantitative reverse transcription-PCR. These results reveal a previously unknown role for cyclic-di-GMP in type 3 fimbrial production.
Replication ofVibrio choleraeChromosome I inEscherichia coli: Dependence on Dam Methylation
Authors: Koch Birgit , Ma Xiaofang , Løbner-Olesen Anders
Published: May 2010

We successfully substitutedEscherichia coli's origin of replicationoriCwith the origin region ofVibrio choleraechromosome I (oriCIVc). Replication fromoriCIVcinitiated at a similar or slightly reduced cell mass compared to that of normalE. coli oriC. With respect to sequestration-dependent synchrony of initiation and stimulation of initiation by the loss of Hda activity, replication initiation fromoriCandoriCIVcwere similar. Since Hda is involved in the conversion of DnaAATP(DnaA bound to ATP) to DnaAADP(DnaA bound to ADP), this indicates that DnaA associated with ATP is limiting forV. choleraechromosome I replication, which similar to what is observed forE. coli. Nohdahomologue has been identified inV. choleraeyet. InV. cholerae,damis essential for viability, whereas inE. coli,dammutants are viable. Replacement ofE. coli oriCwithoriCIVcallowed us to specifically address the role of the Dam methyltransferase and SeqA in replication initiation fromoriCIVc. We show that whenE. coli's origin of replication is substituted byoriCIVc,dam, but notseqA, becomes important for growth, arguing that Dam methylation exerts a critical function at the origin of replication itself. We propose that Dam methylation promotes DnaA-assisted successful duplex opening and replisome assembly atoriCIVcinE. coli. In this model, methylation atoriCIVcwould ease DNA melting. This is supported by the fact that the requirement fordamcan be alleviated by increasing negative supercoiling of the chromosome through oversupply of the DNA gyrase or loss of SeqA activity.
Complete Genome Sequence of Probiotic
Bifidobacterium animalis
subsp.
lactis
Strain V9
Authors: Sun Zhihong , Chen Xia , Wang Jicheng , Gao Pengfei , Zhou Zhemin , Ren Yi , Sun Tiansong , Wang Lei , Meng He , Chen Wei , Zhang Heping
Published: May 2010

Bifidobacterium animalis
subsp.
lactis
strain V9 is a Chinese commercial bifidobacteria with several probiotic functions. It was isolated from a healthy Mongolian child in China. We present here the complete genome sequence of V9 and compare it to 3 other published genome sequences of
B. animalis
subsp.
lactis
strains. The result indicates the lack of polymorphism among strains of this subspecies from different continents.
Nooks and Crannies in Type VI Secretion Regulation
Authors: Bernard Christophe S. , Brunet Yannick R. , Gueguen Erwan , Cascales Eric
Published: May 2010

Type VI secretion systems (T6SS) are macromolecular, transenvelope machines encoded within the genomes of most Gram-negative bacteria, including plant, animal, and human pathogens, as well as soil and environmental isolates. T6SS are involved in a broad variety of functions: from pathogenesis to biofilm formation and stress sensing. This large array of functions is reflected by a vast diversity of regulatory mechanisms: repression by histone-like proteins and regulation by quorum sensing, transcriptional factors, two-component systems, alternative sigma factors, or small regulatory RNAs. Finally, T6SS may be produced in an inactive state and are turned on through the action of a posttranslational cascade involving phosphorylation and subunit recruitment. The current data reviewed here highlight how T6SS have been integrated into existing regulatory networks and how the expression of the T6SS loci is precisely modulated to adapt T6SS production to the specific needs of individual bacteria.
Revisiting the Assignment of Rv0241c to Fatty Acid Synthase Type II ofMycobacterium tuberculosis
Authors: Sacco Emmanuelle , Slama Nawel , Bäckbro Kristina , Parish Tanya , Laval Françoise , Daffé Mamadou , Eynard Nathalie , Quémard Annaık̈
Published: May 2010

The fatty acid synthase type II enzymatic complex ofMycobacterium tuberculosis(FAS-IIMt) catalyzes an essential metabolic pathway involved in the biosynthesis of major envelope lipids, mycolic acids. The partner proteins of this singular FAS-II system represent relevant targets for antituberculous drug design. Two heterodimers of the hydratase 2 protein family, HadAB and HadBC, were shown to be involved in the (3R)-hydroxyacyl-ACP dehydration (HAD) step of FAS-IIMtcycles. Recently, an additional member of this family, Rv0241c, was proposed to have the same function, based on the heterologous complementation of a HAD mutant of the yeast mitochondrial FAS-II system. In the present work,Rv0241cwas able to complement a HAD mutant in theEscherichia colimodel but not a dehydratase-isomerase deficient mutant. However, an enzymatic study of the purified protein demonstrated that Rv0241c possesses a broad chain length specificity for the substrate, unlike FAS-IIMtenzymes. Most importantly, Rv0241c exhibited a strict dependence on the coenzyme A (CoA) as opposed to AcpM, the natural acyl carrier protein bearing the chains elongated by FAS-IIMt. The deletion ofRv0241cshowed that this gene is not essential toM. tuberculosissurvivalin vitro. The resulting mutant did not display any change in the mycolic acid profile. This demonstrates that Rv0241c is atrans-2-enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydratase that does not belong to FAS-IIMt. The relevance of a heterologous complementation strategy to identifying proteins of such a system is questioned.
Role of Multiple HLR1 Sequences in the Regulation of the Dual Promoters of thepsaABGenes inSynechocystissp. PCC 6803
Authors: Takahashi Tomoko , Nakai Nanako , Muramatsu Masayuki , Hihara Yukako
Published: May 2010

Previously, we analyzed the promoter architecture of thepsaABgenes encoding reaction center subunits of photosystem I (PSI) in the cyanobacteriumSynechocystissp. PCC 6803. There exist two promoters, P1 and P2, both of which show typical high-light (HL) response of PSI genes; their activities are high under low-light (LL) conditions but rapidly downregulated upon the shift to HL conditions. In this study, it was suggested that a response regulator RpaB binds to multiple high-light regulatory 1 (HLR1) sequences in the upstream region of thepsaABgenes. We explored the regulatory role ofcis-elements, including these HLR1 sequences on the individual activity of P1 and P2. Under LL conditions, the most influentialcis-element is HLR1C (−62 to −45, relative to the transcriptional starting point of P1) working for positive regulation of P1. The other HLR1 sequences also affect the promoter activity under LL conditions; HLR1A (−255 to −238) is involved in repression of P1, whereas HLR1B (−153 to −126) works for activation of P2. Upon the shift to HL conditions, regulation via HNE2 located within the region from −271 to −177 becomes active in order to downregulate both P1 and P2 activities. A positive effect of HLR1B on P2 may persist under HL. These results suggest thatcis-elements, including multiple HLR1 sequences, differently regulate the activities of dual promoters of thepsaABgenes to achieve the fine-tuning of the gene expression.
Evidence for Widespread Gene Control Function by the
ydaO
Riboswitch Candidate
Authors: Block Kirsten F. , Hammond Ming C. , Breaker Ronald R.
Published: May 2010

Nearly all representatives of experimentally validated riboswitch classes in bacteria control the expression of genes for the transport or synthesis of key metabolic compounds. Recent findings have revealed that some riboswitches also regulate genes involved in physiological changes, virulence, and stress responses. Many novel RNA motifs are being identified by using bioinformatics algorithms that search for conserved sequence and structural features located in intergenic regions. Some of these RNAs are likely to function as riboswitches for metabolites or signaling compounds, and confirmation of this function would reveal the basis of the genetic control of new regulons. Herein we describe the analysis of the
ydaO
riboswitch candidate, which represents one of the most widespread candidates remaining to be validated. These RNAs are common in Gram-positive bacteria, and their genomic associations with diverse genes suggest that they sense a compound that signals broader physiological changes. We determined that the
ydaO
motif exhibits sequence- and structure-dependent gene control, and reporter assays indicate that its natural ligand is present even when cells are grown in defined media. A transposon-mediated knockout screen resulted in mutants with a dysregulated expression of genes controlled by the RNA motif. The mutations disrupt genes that drastically modulate energy-generating pathways, suggesting that the intracellular concentration of the ligand sensed by the
ydaO
motif is altered under these stress conditions.
The Threshold Level of the Sensor Histidine Kinase KinA Governs Entry into Sporulation in
Bacillus subtilis
Authors: Eswaramoorthy Prahathees , Duan Daniel , Dinh Jeffrey , Dravis Ashlee , Devi Seram Nganbiton , Fujita Masaya
Published: May 2010

Sporulation in
Bacillus subtilis
is controlled by a complex gene regulatory circuit that is activated upon nutrient deprivation. The initial process is directed by the phosphorelay, involving the major sporulation histidine kinase (KinA) and two additional phosphotransferases (Spo0F and Spo0B), that activates the master transcription factor Spo0A. Little is known about the initial event and mechanisms that trigger sporulation. Using a strain in which the synthesis of KinA is under the control of an IPTG (isopropyl-β-
d
-thiogalactopyranoside)-inducible promoter, here we demonstrate that inducing the synthesis of the KinA beyond a certain level leads to the entry of the irreversible process of sporulation irrespective of nutrient availability. Moreover, the engineered cells expressing KinA under a σ
H
-dependent promoter that is similar to but stronger than the endogenous
kinA
promoter induce sporulation during growth. These cells, which we designated COS (
co
nstitutive
s
porulation) cells, exhibit the morphology and properties of sporulating cells and express sporulation marker genes under nutrient-rich conditions. Thus, we created an engineered strain displaying two cell cycles (growth and sporulation) integrated into one cycle irrespective of culture conditions, while in the wild type, the appropriate cell fate decision is made depending on nutrient availability. These results suggest that the threshold level of the major sporulation kinase acts as a molecular switch to determine cell fate and may rule out the possibility that the activity of KinA is regulated in response to the unknown signal(s).
Mutations Affecting Export and Activity of Cytolysin A from
Escherichia coli
Authors: Ludwig Albrecht , Völkerink Guido , von Rhein Christine , Bauer Susanne , Maier Elke , Bergmann Birgit , Goebel Werner , Benz Roland
Published: May 2010

Cytolysin A (known as ClyA, HlyE, and SheA) is a cytolytic pore-forming protein toxin found in several
Escherichia coli
and
Salmonella enterica
strains. The structure of its water-soluble monomeric form and that of dodecameric ClyA pores is known, but the mechanisms of ClyA export from bacterial cells and of pore assembly are only partially understood. Here we used site-directed mutagenesis to study the importance of different regions of the
E. coli
ClyA protein for export and activity. The data indicate that ClyA translocation to the periplasm requires several protein segments located closely adjacent to each other in the “tail” domain of the ClyA monomer, namely, the N- and C-terminal regions and the hydrophobic sequence ranging from residues 89 to 101. Deletion of most of the “head” domain of the monomer (residues 181 to 203), on the other hand, did not strongly affect ClyA secretion, suggesting that the tail domain plays a particular role in export. Furthermore, we found that the N-terminal amphipathic helix αA1 of ClyA is crucial for the formation and the properties of the transmembrane channel, and hence for hemolytic activity. Several mutations affecting the C-terminal helix αG, the “β-tongue” region in the head domain, or the hydrophobic region in the tail domain of the ClyA monomer strongly impaired the hemolytic activity and reduced the activity toward planar lipid bilayer membranes but did not totally prevent formation of wild-type-like channels in these artificial membranes. The latter regions thus apparently promote membrane interaction without being directly required for pore formation in a lipid bilayer.