bluuebunny/crossref_metadata_2025 huggingface dataset:
The influence of housefly Musca domestica embryo age on viability in water and at low temperatures
Authors: ČIČKOVÁ H. , KOZÁNEK M. , TAKÁČ P.
Published: August 2013

The sensitivity of housefly Musca domestica L. (Diptera: Muscidae) embryos to storage at low temperatures (5 and 10 °C on moist sponges in Petri dishes) and in water at 26 °C was investigated to develop suitable protocols for the storage and transport of housefly eggs. The youngest embryos (aged 0–3 h) were the most sensitive to storage at 5 °C, with 45% survival after storage for 24 h. Storage of embryos aged 3–12 h at 5 °C for 24 h had no negative effect; longer storage resulted in significantly decreased larval survival (30–34% after 48–72 h, compared with 61% in the control group) and reduced hatching rates (83% after 72 h storage). No negative effects were observed when embryos aged 0–9 h were stored at 10 °C for 24 h, but this temperature did not completely inhibit development and eggs began to hatch if stored for longer than 24 h. All age groups of embryos showed high mortality after storage in water at 26 °C for 24 h, with the youngest embryos being least resistant to submersion.
New postcranial elements of the Thalassodrominae (Pterodactyloidea, Tapejaridae) from the Romualdo Formation (Aptian–Albian), Santana Group, Araripe Basin, Brazil
Authors: Aires Alex S. S. , Kellner Alexander W. A. , Müller Rodrigo T. , Da Silva Lúcio R. , Pacheco Cristian P. , Dias‐Da‐Silva Sérgio
Published: August 2013

Tapejarids are edentate pterosaurs recovered mainly from Early Cretaceous deposits. They are diagnosed by five synapomorphies, among which only one is postcranial: a broad and well‐developed tubercle at the ventroposterior margin of the coracoid. Regarding the clade Thalassodrominae, most phylogenetic studies are based on cranial elements, as postcranial skeletons of these pterosaurs are rare. Here, new postcranial material from the Romualdo Formation (Aptian–Albian) from the Araripe Basin is described. The material comprises the three posteriormost cervical vertebrae, the first seven dorsal vertebrae (fused into a notarium), both scapulocoracoids, a fragment of a sternum, a partial right humerus, a small fragment of a 4th phalanx of the wing finger, a distal extremity of the right femur and the proximal portions of both tibia and fibula. Comparisons with other specimens and morphological features examined in a phylogenetic context, such as the presence of three foramina lateral and dorsal to the neural canal of the cervical vertebrae, the presence of a notarium and a pneumatic foramen on the ventral side of the proximal portion of the humerus, allow the assignment of this specimen as Thalassodrominae indet. Regarding palaeobiogeographical aspects, to date, this clade is exclusively found in the Romualdo Formation. It is the most complete postcranial material assigned to the Thalassodrominae described so far.
Neonatal pleural empyema caused by emm type 6 group A streptococcus
Authors: Nohara Fumikatsu , Nagaya Ken , Asai Hiroko , Tsuchida Etsushi , Okamoto Toshio , Hayashi Tokitsugi , Sakata Hiroshi , Terao Yutaka , Azuma Hiroshi
Published: August 2013

Since the mid‐1980s, there have been increasing reports of severe invasive group A streptococcal (GAS) disease in children and adults. There are few reports, however, of neonatal invasive disease, particularly neonatal pleural empyema caused by GAS. Although many mechanisms have been reported for the pathophysiology of invasive GAS infections, similar reports for neonates were unable to be located. Reported herein is the case of a 3‐day‐old girl with pleural empyema caused by GAS that demonstrated a high invasive capacity for human epithelial cells.
Episodic idiopathic systemic capillary leak syndrome in a girl
Authors: Milani Gregorio Paolo , Dellepiane Rosa Maria , Castellazzi Massimo Luca , Mazzoni Marta Benedetta Maria , Bianchetti Mario Giovanni , Fossali Emilio Filippo
Published: August 2013

Episodic idiopathic systemic capillary leak syndrome is a rare disorder that presents with attacks of circulatory shock due to plasma leakage into the extravascular space. Reported here is the case of a girl who had recurrent circulatory shock. The diagnosis of episodic idiopathic systemic capillary leak syndrome was made following the fourth episode. The course was favorable following acute i.v. cristalloids and methylprednisolone.
Case of an infant with hepatic cirrhosis caused by mitochondrial respiratory chain disorder
Authors: Enkai Shigehiro , Koinuma Sachi , Ito Reiko , Igaki Junko , Hasegawa Yukihiro , Murayama Kei , Ohtake Akira
Published: August 2013

The patient had hepatomegaly with liver dysfunction at the age of 1 month. Magnetic resonance imaging performed at the age of 1 year showed multiple nodules of varying size in his liver. We were able to examine the mitochondrial respiratory chain function in the liver biopsy samples because all other differential diagnoses for hepatic cirrhosis had been ruled out. Complex I and IV activities were below the normal level (<30%) of the citrate synthase (CS) ratio. Liver blue native polyacrylamide gel electrophoresis showed an extremely weak complex I and IV band. Liver respiratory chain complexes I and IV were found to be deficient in this patient. The histologic findings were highly suggestive of mitochondrial respiratory chain disorder. Findings of progressive liver cirrhosis changes were observed in magnetic resonance imaging at the age of 5 years. An examination of the mitochondrial respiratory chain function should be performed along with a liver biopsy if mitochondrial respiratory chain disorder is suspected as a possible differential diagnosis of idiopathic hepatitis.
Anaphylactic reaction to polyethylene‐glycol conjugated‐asparaginase: Premedication and desensitization may not be sufficient
Authors: Sahiner Umit M. , Yavuz S. Tolga , Gökce Muge , Buyuktiryaki Betul , Altan Ilhan , Aytac Selin , Tuncer Murat , Tuncer Ayfer , Sackesen Cansin
Published: August 2013

In hypersensitive reactions to native L‐asparaginase, either premedication and desensitization or substitution with polyethylene glycol conjugated asparaginase (PEG‐ASP) is preferred. Anaphylaxis with PEG‐ASP is rare. An 8‐year‐old girl and a 2.5‐year‐old boy, both diagnosed as having acute lymphoblastic leukemia, presented with native L‐asparaginase hypersensitivity and substitution with PEG‐ASP was preferred. They received a premedication (methylprednisolone, hydroxyzine and ranitidine) followed by desensitization with PEG‐ASP infusion. Both patients developed anaphylaxis with peg‐asparaginase. These are the first reported cases of anaphylactic reaction to PEG‐ASP, despite the application of both premedication and desensitization. Anaphylaxis with PEG‐ASP is very rare and premedication and desensitization protocols may not prevent these hypersensitive reactions.
Photooxidation Mechanism of Levomepromazine in Different Solvents
Authors: Piñero‐Santiago Luis E. , García Carmelo , Lhiaubet‐Vallet Virginie , Trzcionka Jerome , Oyola Rolando , Torres Karen , Leguillú Jaysika , Miranda Miguel A.
Published: August 2013

Unwanted photoinduced responses are well‐known adverse effects of most promazine drugs, including levomepromazine (LPZ, Levoprome® or Nozinan®). This drug is indicated in psychiatry primarily for the treatment of schizophrenia and other schizoaffective disorders. Levomepromazine's particular sedative properties make it especially fit for use in psychiatric intensive care. Nevertheless, it is photolabile under UV‐A and UV‐B light in aerobic conditions resulting in the formation of its sulfoxide. The LPZ photochemistry in acetonitrile (MeCN) is completely different from that in methanol (MeOH) and phosphate buffer solutions (PBS, pH = 7.4). The major photoproduct in PBS and MeOH under aerobic conditions is levomepromazine sulfoxide (LPZSO), although the amount is considerably higher in the aqueous environment. The corresponding main photoproduct in MeCN could not be characterized. The destruction quantum yields of LPZ in PBS, MeOH and MeCN are 0.13, 0.02 and <10−3, respectively. It is further demonstrated that LPZSO does not form by the reaction of singlet oxygen with ground‐state LPZ. This oxidation product is actually produced by the reaction of the cation radical of LPZ (LPZ·+) with molecular oxygen. This cation radical in turn, is produced by an electron transfer process between the 3LPZ* and ground‐state molecular oxygen.
A Framework for Understanding Risk Perception, Explored from the Perspective of the Water Practitioner
Authors: Dobbie Meredith Frances , Brown Rebekah Ruth
Published: August 2013

Sustainable urban water systems are likely to be hybrids of centralized and decentralized infrastructure, managed as an integrated system in water‐sensitive cities. The technology for many of these systems is available. However, social and institutional barriers, which can be understood as deeply embedded risk perceptions, have impeded their implementation. Risk perceptions within the water sector are often unrecognized or unacknowledged, despite their role in risk management generally in informing value judgments and specifically in ranking risks to achieve management objectives. There has been very little examination of the role of these risk perceptions in advancing more sustainable water supply management through the adoption of alternative sources. To address this gap, this article presents a framework that can be used as a tool for understanding risk perceptions. The framework is built on the relational theory of risk and presents the range of human phenomena that might influence the perception of an “object at risk” in relation to a “risk object.” It has been synthesized from a critical review of theoretical, conceptual, and empirical studies of perception broadly and risk perception specifically, and interpreted in relation to water practitioners. For a water practitioner, the risk object might be an alternative water system, a component, a process, or a technology, and the object at risk could be public or environmental health, profitability, or professional reputation. This framework has two important functions: to allow practitioners to understand their own and others’ risk perceptions, which might differ, and to inform further empirical research.
Elucidating the temporal and spatial dynamics of Biomphalaria glabrata genetic diversity in three Brazilian villages
Authors: Thiele Elizabeth A. , Corrêa‐Oliveira Guilherme , Gazzinelli Andrea , Minchella Dennis J.
Published: August 2013

Objective

The freshwater snail Biomphalaria glabrata is the principal intermediate host for the parasite Schistosoma mansoni within Brazil. We assessed the potential effects of snail population dynamics on parasite transmission dynamics via population genetics.

Methods

We sampled snail populations located within the confines of three schistosome‐endemic villages in the state of Minas Gerais, Brazil. Snails were collected from individual microhabitats following seasonal periods of flood and drought over the span of 1 year. Snail spatio‐temporal genetic diversity and population differentiation of 598 snails from 12 sites were assessed at seven microsatellite loci.

Results

Average genetic diversity was relatively low, ranging from 4.29 to 9.43 alleles per locus, and overall, subpopulations tended to exhibit heterozygote deficits. Genetic diversity was highly spatially partitioned among subpopulations, while virtually, no partitioning was observed across temporal sampling. Comparison with previously published parasite genetic diversity data indicated that S. mansoni populations are significantly more variable and less subdivided than those of the B. glabrata intermediate hosts.

Discussion

Within individual Brazilian villages, observed distributions of snail genetic diversity indicate temporal stability and very restricted gene flow. This is contrary to observations of schistosome genetic diversity over the same spatial scale, corroborating the expectation that parasite gene flow at the level of individual villages is likely driven by vertebrate host movement.
Model selection reveals control of cold signalling by evening‐phased components of the plant circadian clock
Authors: Keily Jack , MacGregor Dana R. , Smith Robert W. , Millar Andrew J. , Halliday Karen J. , Penfield Steven
Published: August 2013

Circadian clocks confer advantages by restricting biological processes to certain times of day through the control of specific phased outputs. Control of temperature signalling is an important function of the plant oscillator, but the architecture of the gene network controlling cold signalling by the clock is not well understood. Here we use a model ensemble fitted to time‐series data and a corrected Akaike Information Criterion (AICc) analysis to extend a dynamic model to include the control of the key cold‐regulated transcription factors C‐REPEAT BINDING FACTORs 1–3 (CBF1, CBF2, CBF3). AICc was combined with in silico analysis of genetic perturbations in the model ensemble, and selected a model that predicted mutant phenotypes and connections between evening‐phased circadian clock components and CBF3 transcriptional control, but these connections were not shared by CBF1 and CBF2. In addition, our model predicted the correct gating of CBF transcription by cold only when the cold signal originated from the clock mechanism itself, suggesting that the clock has an important role in temperature signal transduction. Our data shows that model selection could be a useful method for the expansion of gene network models.
Role of oxidative stress‐induced endothelin‐converting enzyme activity in the alteration of carotid body function by chronic intermittent hypoxia
Authors: Peng Ying‐Jie , Nanduri Jayasri , Raghuraman Gayatri , Wang Ning , Kumar Ganesh K. , Prabhakar Nanduri R.
Published: August 2013

New findings

What is the central question of this study? 

What mechanisms mediate chronic intermittent hypoxia‐induced increases in endothelin‐1 in the carotid body?

What is the main finding and its importance? 

Upregulation of endothelin‐1 by chronic intermittent hypoxia results from reactive oxygen species‐dependent activation of endothelin‐converting enzyme and is not the result of augmented endothelin‐1 gene transcriptional activity. The resultant increased endothelin‐1 acts via endothelin‐1A receptors to induce hypoxic hypersensitivity of the carotid body but does not contribute to the sensory long‐term facilitation observed in this condition. These findings provide mechanistic insight into how chronic intermittent hypoxia alters carotid body function.

Chronic intermittent hypoxia (CIH) leads to remodelling of the carotid body function, manifested by an augmented sensory response to hypoxia and induction of sensory long‐term facilitation (LTF). It was proposed that endothelin‐1 (ET‐1) contributes to CIH‐induced hypoxic hypersensitivity of the carotid body. The objectives of the present study were as follows: (i) to delineate the mechanisms by which CIH upregulates ET‐1 expression in the carotid body; and (ii) to assess whether ET‐1 also contributes to sensory LTF. Experiments were performed on adult, male rats exposed to alternating cycles of 5% O2 (15 s) and room air (5 min), nine episodes per hour and 8 h per day for 10 days. Chronic intermittent hypoxia increased ET‐1 levels in glomus cells without significantly altering prepro‐endothelin‐1 mRNA levels. The activity of endothelin‐converting enzyme increased with concomitant elevation of ET‐1 levels in CIH‐exposed carotid bodies, and MnTMPyP, a membrane‐permeable antioxidant, prevented these effects. Hypoxia facilitated ET‐1 release from CIH‐treated carotid bodies, which is a prerequisite for activation of ET receptors; however, hypoxia had no effect on ET‐1 release from control carotid bodies. In CIH‐exposed carotid bodies, mRNAs encoding ETA receptor were upregulated, and an ETA receptor‐specific antagonist abolished CIH‐induced hypersensitivity of the hypoxic response, whereas it had no effect on the sensory LTF. These results suggest that ECE‐dependent increased production of ET‐1 coupled with hypoxia‐evoked ET‐1 release and the ensuing ETA receptor activation mediate the CIH‐induced carotid body hypersensitivity to hypoxia, but the ETA signalling pathway is not associated with sensory LTF elicited by CIH.
Coherence among Different Microbial Source Tracking Markers in a Small Agricultural Stream with or without Livestock Exclusion Practices
Authors: Wilkes Graham , Brassard Julie , Edge Thomas A. , Gannon Victor , Jokinen Cassandra C. , Jones Tineke H. , Marti Romain , Neumann Norman F. , Ruecker Norma J. , Sunohara Mark , Topp Edward , Lapen David R.
Published: August 2013

Over 1,400 water samples were collected biweekly over 6 years from an intermittent stream protected and unprotected from pasturing cattle. The samples were monitored for host-specific
Bacteroidales
markers,
Cryptosporidium
species/genotypes, viruses and coliphages associated with humans or animals, and bacterial zoonotic pathogens. Ruminant
Bacteroidales
markers did not increase within the restricted cattle access reach of the stream, whereas the ruminant
Bacteroidales
marker increased significantly in the unrestricted cattle access reach. Human
Bacteroidales
markers significantly increased downstream of homes where septic issues were documented. Wildlife
Bacteroidales
markers were detected downstream of the cattle exclusion practice where stream and riparian habitat was protected, but detections decreased after the unrestricted pasture, where the stream and riparian zone was unprotected from livestock. Detection of a large number of human viruses was shown to increase downstream of homes, and similar trends were observed for the human
Bacteroidales
marker. There was considerable interplay among biomarkers with stream flow, season, and the cattle exclusion practices. There were no to very weak associations with
Bacteroidales
markers and bacterial, viral, and parasitic pathogens. Overall, discrete sample-by-sample coherence among the different microbial source tracking markers that expressed a similar microbial source was minimal, but spatial trends were physically meaningful in terms of land use (e.g., beneficial management practice) effects on sources of fecal pollution.
Metagenome Survey of a Multispecies and Alga-Associated Biofilm Revealed Key Elements of Bacterial-Algal Interactions in Photobioreactors
Authors: Krohn-Molt Ines , Wemheuer Bernd , Alawi Malik , Poehlein Anja , Güllert Simon , Schmeisser Christel , Pommerening-Röser Andreas , Grundhoff Adam , Daniel Rolf , Hanelt Dieter , Streit Wolfgang R.
Published: August 2013

Photobioreactors (PBRs) are very attractive for sunlight-driven production of biofuels and capturing of anthropogenic CO
2
. One major problem associated with PBRs however, is that the bacteria usually associated with microalgae in nonaxenic cultures can lead to biofouling and thereby affect algal productivity. Here, we report on a phylogenetic, metagenome, and functional analysis of a mixed-species bacterial biofilm associated with the microalgae
Chlorella vulgaris
and
Scenedesmus obliquus
in a PBR. The biofilm diversity and population dynamics were examined through 16S rRNA phylogeny. Overall, the diversity was rather limited, with approximately 30 bacterial species associated with the algae. The majority of the observed microorganisms were affiliated with
Alphaproteobacteria
,
Betaproteobacteria
, and
Bacteroidetes
. A combined approach of sequencing via GS FLX Titanium from Roche and HiSeq 2000 from Illumina resulted in the overall production of 350 Mbp of sequenced DNA, 165 Mbp of which was assembled in larger contigs with a maximum size of 0.2 Mbp. A KEGG pathway analysis suggested high metabolic diversity with respect to the use of polymers and aromatic and nonaromatic compounds. Genes associated with the biosynthesis of essential B vitamins were highly redundant and functional. Moreover, a relatively high number of predicted and functional lipase and esterase genes indicated that the alga-associated bacteria are possibly a major sink for lipids and fatty acids produced by the microalgae. This is the first metagenome study of microalga- and PBR-associated biofilm bacteria, and it gives new clues for improved biofuel production in PBRs.
ClpB1 Overproduction in Synechocystis sp. Strain PCC 6803 Increases Tolerance to Rapid Heat Shock
Authors: Gonzalez-Esquer C. Raul , Vermaas Wim F. J.
Published: August 2013

ClpB1 is a heat shock protein known to disaggregate large protein complexes. Constitutive, 16-fold ClpB1 overproduction in the cyanobacterium
Synechocystis
sp. strain PCC 6803 increased cell survival by 20-fold when cultures were heated quickly (1°C/s) to 50°C and delayed cell death by an average of 3 min during incubation at high temperatures (>46°C). Cooverexpression of ClpB1 and another heat shock protein, DnaK2, further increased cell survival. According to immunocytochemistry results, ClpB1 is dispersed throughout the cytoplasm but is concentrated in specific areas and is more prevalent near thylakoid membranes. However, ClpB1 overproduction does not lead to a change in the morphology, chlorophyll content, or photosystem ratio. Whereas electron microscopy demonstrated that apparent protein aggregation occurred after heat treatment in the control strain, protein aggregate size was maintained in the ClpB1 overexpresser. Constitutive ClpB1 overproduction allows an earlier response to heat shock and protects from rapid heating of cultures.
Proteomic Analysis of the Quorum-Sensing Regulon in Pantoea stewartii and Identification of Direct Targets of EsaR
Authors: Ramachandran Revathy , Stevens Ann M.
Published: August 2013

The proteobacterium
Pantoea stewartii
subsp.
stewartii
causes Stewart's wilt disease in maize when it colonizes the xylem and secretes large amounts of stewartan, an exopolysaccharide. The success of disease pathogenesis lies in the timing of bacterial virulence factor expression through the different stages of infection. Regulation is achieved through a quorum-sensing (QS) system consisting of the acyl-homoserine lactone (AHL) synthase, EsaI, and the transcription regulator EsaR. At low cell densities, EsaR represses transcription of itself and of
rcsA
, an activator of the stewartan biosynthesis operon; it also activates
esaS
, which encodes a small RNA (sRNA). Repression or activation ceases at high cell densities when EsaI synthesizes sufficient levels of the AHL ligand
N
-3-oxo-hexanoyl-
l
-homoserine lactone to bind and inactivate EsaR. This study aims to identify other genes activated or repressed by EsaR during the QS response. Proteomic analysis identified a QS regulon of more than 30 proteins. Electrophoretic mobility shift assays of promoters of genes encoding differentially expressed proteins distinguished direct targets of EsaR from indirect targets. Additional quantitative reverse transcription-PCR (qRT-PCR) and DNA footprinting analysis established that EsaR directly regulates the promoters of
dkgA
,
glpF
, and
lrhA
. The proteins encoded by
dkgA
,
glpF
, and
lrhA
are a 2,5-diketogluconate reductase, glycerol facilitator, and transcriptional regulator of chemotaxis and motility, respectively, indicating a more global QS response in
P. stewartii
than previously recognized.
Exploring the Biosynthesis of Unsaturated Fatty Acids in Bacillus cereus ATCC 14579 and Functional Characterization of Novel Acyl-Lipid Desaturases
Authors: Chazarreta Cifré Lorena , Alemany Mariana , de Mendoza Diego , Altabe Silvia
Published: August 2013

At low temperatures,
Bacillus cereus
synthesizes large amounts of unsaturated fatty acids (UFAs) with double bonds in positions Δ5 and Δ10, as well as Δ5,10 diunsaturated fatty acids. Through sequence homology searches, we identified two open reading frames (ORFs) encoding a putative Δ5 desaturase and a fatty acid acyl-lipid desaturase in the
B. cereus
ATCC 14579 genome, and these were named BC2983 and BC0400, respectively. Functional characterization of ORFs BC2983 and BC0400 by means of heterologous expression in
Bacillus subtilis
confirmed that they both encode acyl-lipid desaturases that use phospholipids as the substrates and have Δ5 and Δ10 desaturase activities. Thus, these ORFs were correspondingly named
desA
(Δ5 desaturase) and
desB
(Δ10 desaturase). We established that DesA utilizes ferredoxin and flavodoxins (Flds) as electron donors for the desaturation reaction, while DesB preferably employs Flds. In addition, increased amounts of UFAs were found when
B. subtilis
expressing
B. cereus
desaturases was subjected to a cold shock treatment, indicating that the activity or the expression of these enzymes is upregulated in response to a decrease in growth temperature. This represents the first work reporting the functional characterization of fatty acid desaturases from
B. cereus
.
Directed Evolution and Structural Analysis of NADPH-Dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) Reductase from Ralstonia eutropha Reveals Two Mutations Responsible for Enhanced Kinetics
Authors: Matsumoto Ken'ichiro , Tanaka Yoshikazu , Watanabe Tsuyoshi , Motohashi Ren , Ikeda Koji , Tobitani Kota , Yao Min , Tanaka Isao , Taguchi Seiichi
Published: August 2013

NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with β-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from
Ralstonia eutropha
was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based
in vivo
screening system using
Escherichia coli
. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited
k
cat
values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant
Corynebacterium glutamicum
. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.
Metabolism of Four α-Glycosidic Linkage-Containing Oligosaccharides by Bifidobacterium breve UCC2003
Authors: O'Connell Kerry Joan , O'Connell Motherway Mary , O'Callaghan John , Fitzgerald Gerald F. , Ross R. Paul , Ventura Marco , Stanton Catherine , van Sinderen Douwe
Published: August 2013

Members of the genus
Bifidobacterium
are common inhabitants of the gastrointestinal tracts of humans and other mammals, where they ferment many diet-derived carbohydrates that cannot be digested by their hosts. To extend our understanding of bifidobacterial carbohydrate utilization, we investigated the molecular mechanisms by which 11 strains of
Bifidobacterium breve
metabolize four distinct α-glucose- and/or α-galactose-containing oligosaccharides, namely, raffinose, stachyose, melibiose, and melezitose. Here we demonstrate that all
B. breve
strains examined possess the ability to utilize raffinose, stachyose, and melibiose. However, the ability to metabolize melezitose was not common to all
B. breve
strains tested. Transcriptomic and functional genomic approaches identified a gene cluster dedicated to the metabolism of α-galactose-containing carbohydrates, while an adjacent gene cluster, dedicated to the metabolism of α-glucose-containing melezitose, was identified in strains that are able to use this carbohydrate.
Raman Spectroscopy and Chemometrics for Identification and Strain Discrimination of the Wine Spoilage Yeasts Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Brettanomyces bruxellensis
Authors: Rodriguez Susan B. , Thornton Mark A. , Thornton Roy J.
Published: August 2013

The yeastsZygosaccharomyces bailii,Dekkera bruxellensis(anamorph,Brettanomyces bruxellensis), andSaccharomyces cerevisiaeare the major spoilage agents of finished wine. A novel method using Raman spectroscopy in combination with a chemometric classification tool has been developed for the identification of these yeast species and for strain discrimination of these yeasts. Raman spectra were collected for six strains of each of the yeastsZ. bailii,B. bruxellensis, andS. cerevisiae. The yeasts were classified with high sensitivity at the species level: 93.8% forZ. bailii, 92.3% forB. bruxellensis, and 98.6% forS. cerevisiae. Furthermore, we have demonstrated that it is possible to discriminate between strains of these species. These yeasts were classified at the strain level with an overall accuracy of 81.8%.
Oxidative Stress at High Temperatures in Lactococcus lactis Due to an Insufficient Supply of Riboflavin
Authors: Chen Jun , Shen Jing , Solem Christian , Jensen Peter Ruhdal
Published: August 2013

Lactococcus lactis
MG1363 was found to be unable to grow at temperatures above 37°C in a defined medium without riboflavin, and the cause was identified to be dissolved oxygen introduced during preparation of the medium. At 30°C, growth was unaffected by dissolved oxygen and oxygen was consumed quickly. Raising the temperature to 37°C resulted in severe growth inhibition and only slow removal of dissolved oxygen. Under these conditions, an abnormally low intracellular ratio of [ATP] to [ADP] (1.4) was found (normally around 5), which indicates that the cells are energy limited. By adding riboflavin to the medium, it was possible to improve growth and oxygen consumption at 37°C, and this also normalized the [ATP]-to-[ADP] ratio. A codon-optimized redox-sensitive green fluorescent protein (GFP) was introduced into
L. lactis
and revealed a more oxidized cytoplasm at 37°C than at 30°C. These results indicate that
L. lactis
suffers from heat-induced oxidative stress at increased temperatures. A decrease in intracellular flavin adenine dinucleotide (FAD), which is derived from riboflavin, was observed with increasing growth temperature, but the presence of riboflavin made the decrease smaller. The drop was accompanied by a decrease in NADH oxidase and pyruvate dehydrogenase activities, both of which depend on FAD as a cofactor. By overexpressing the riboflavin transporter, it was possible to improve FAD biosynthesis, which resulted in increased NADH oxidase and pyruvate dehydrogenase activities and improved fitness at high temperatures in the presence of oxygen.
The Virulence of Salmonella enterica Serovar Typhimurium in the Insect Model Galleria mellonella Is Impaired by Mutations in RNase E and RNase III
Authors: Viegas Sandra C. , Mil-Homens Dalila , Fialho Arsénio M. , Arraiano Cecília M.
Published: August 2013

Salmonella enterica
serovar Typhimurium is a Gram-negative bacterium able to invade and replicate inside eukaryotic cells. To cope with the host defense mechanisms, the bacterium has to rapidly remodel its transcriptional status. Regulatory RNAs and ribonucleases are the factors that ultimately control the fate of mRNAs and final protein levels in the cell. There is growing evidence of the direct involvement of these factors in bacterial pathogenicity. In this report, we validate the use of a
Galleria mellonela
model in
S
. Typhimurium pathogenicity studies through the parallel analysis of a mutant with a mutation in
hfq
, a well-established
Salmonella
virulence gene. The results obtained with this mutant are similar to the ones reported in a mouse model. Through the use of this insect model, we demonstrate a role for the main endoribonucleases RNase E and RNase III in
Salmonella
virulence. These ribonuclease mutants show an attenuated virulence phenotype, impairment in motility, and reduced proliferation inside the host. Interestingly, the two mutants trigger a distinct immune response in the host, and the two mutations seem to have an impact on distinct bacterial functions.
Carbonylation as a Key Reaction in Anaerobic Acetone Activation by Desulfococcus biacutus
Authors: Gutiérrez Acosta Olga B. , Hardt Norman , Schink Bernhard
Published: August 2013

Acetone is activated by aerobic and nitrate-reducing bacteria via an ATP-dependent carboxylation reaction to form acetoacetate as the first reaction product. In the activation of acetone by sulfate-reducing bacteria, acetoacetate has not been found to be an intermediate. Here, we present evidence of a carbonylation reaction as the initial step in the activation of acetone by the strictly anaerobic sulfate reducer
Desulfococcus biacutus
. In cell suspension experiments, CO was found to be a far better cosubstrate for acetone activation than CO
2
. The hypothetical reaction product, acetoacetaldehyde, is extremely reactive and could not be identified as a free intermediate. However, acetoacetaldehyde dinitrophenylhydrazone was detected by mass spectrometry in cell extract experiments as a reaction product of acetone, CO, and dinitrophenylhydrazine. In a similar assay, 2-amino-4-methylpyrimidine was formed as the product of a reaction between acetoacetaldehyde and guanidine. The reaction depended on ATP as a cosubstrate. Moreover, the specific activity of aldehyde dehydrogenase (coenzyme A [CoA] acylating) tested with the putative physiological substrate was found to be 153 ± 36 mU mg
−1
protein, and its activity was specifically induced in extracts of acetone-grown cells. Moreover, acetoacetyl-CoA was detected (by mass spectrometry) after the carbonylation reaction as the subsequent intermediate after acetoacetaldehyde was formed. These results together provide evidence that acetoacetaldehyde is an intermediate in the activation of acetone by sulfate-reducing bacteria.
Candida albicans
VMA3
Is Necessary for V-ATPase Assembly and Function and Contributes to Secretion and Filamentation
Authors: Rane Hallie S. , Bernardo Stella M. , Raines Summer M. , Binder Jessica L. , Parra Karlett J. , Lee Samuel A.
Published: August 2013

The vacuolar membrane ATPase (V-ATPase) is a protein complex that utilizes ATP hydrolysis to drive protons from the cytosol into the vacuolar lumen, acidifying the vacuole and modulating several key cellular response systems in
Saccharomyces cerevisiae
. To study the contribution of V-ATPase to the biology and virulence attributes of the opportunistic fungal pathogen
Candida albicans
, we created a conditional mutant in which
VMA3
was placed under the control of a tetracycline-regulated promoter (tetR-
VMA3
strain). Repression of
VMA3
in the tetR-
VMA3
strain prevents V-ATPase assembly at the vacuolar membrane and reduces concanamycin A-sensitive ATPase-specific activity and proton transport by more than 90%. Loss of
C. albicans
V-ATPase activity alkalinizes the vacuolar lumen and has pleiotropic effects, including pH-dependent growth, calcium sensitivity, and cold sensitivity. The tetR-
VMA3
strain also displays abnormal vacuolar morphology, indicative of defective vacuolar membrane fission. The tetR-
VMA3
strain has impaired aspartyl protease and lipase secretion, as well as attenuated virulence in an
in vitro
macrophage killing model. Repression of
VMA3
suppresses filamentation, and V-ATPase-dependent filamentation defects are not rescued by overexpression of
RIM8
,
MDS3
,
EFG1
,
CST20
, or
UME6
, which encode positive regulators of filamentation. Specific chemical inhibition of Vma3p function also results in defective filamentation. These findings suggest either that V-ATPase functions downstream of these transcriptional regulators or that V-ATPase function during filamentation involves independent mechanisms and alternative signaling pathways. Taken together, these data indicate that V-ATPase activity is a fundamental requirement for several key virulence-associated traits in
C. albicans
.
Comparative Evolution of Morphological Regulatory Functions in Candida Species
Authors: Lackey Erika , Vipulanandan Geethanjali , Childers Delma S. , Kadosh David
Published: August 2013

Morphological transitions play an important role in virulence and virulence-related processes in a wide variety of pathogenic fungi, including the most commonly isolated human fungal pathogen
Candida albicans
. While environmental signals, transcriptional regulators, and target genes associated with
C. albicans
morphogenesis are well-characterized, considerably little is known about morphological regulatory mechanisms and the extent to which they are evolutionarily conserved in less pathogenic and less filamentous non-
albicans Candida
species (NACS). We have identified specific optimal filament-inducing conditions for three NACS (
C. tropicalis
,
C. parapsilosis
, and
C. guilliermondii
), which are very limited, suggesting that these species may be adapted for niche-specific filamentation in the host. Only a subset of evolutionarily conserved
C. albicans
filament-specific target genes were induced upon filamentation in
C. tropicalis
,
C. parapsilosis
, and
C. guilliermondii
. One of the genes showing conserved expression was
UME6
, a key filament-specific regulator of
C. albicans
hyphal development. Constitutive high-level expression of
UME6
was sufficient to drive increased filamentation as well as biofilm formation and partly restore conserved filament-specific gene expression in both
C. tropicalis
and
C. parapsilosis
, suggesting that evolutionary differences in filamentation ability among pathogenic
Candida
species may be partially attributed to alterations in the expression level of a conserved filamentous growth machinery. In contrast to
UME6
,
NRG1
, an important repressor of
C. albicans
filamentation, showed only a partly conserved role in controlling NACS filamentation. Overall, our results suggest that
C. albicans
morphological regulatory functions are partially conserved in NACS and have evolved to respond to more specific sets of host environmental cues.
Identification of the Set of Genes, Including Nonannotated
morA
, under the Direct Control of ModE in Escherichia coli
Authors: Kurata Tatsuaki , Katayama Akira , Hiramatsu Masakazu , Kiguchi Yuya , Takeuchi Masamitsu , Watanabe Tomoyuki , Ogasawara Hiroshi , Ishihama Akira , Yamamoto Kaneyoshi
Published: August 2013

ModE is the molybdate-sensing transcription regulator that controls the expression of genes related to molybdate homeostasis in
Escherichia coli
. ModE is activated by binding molybdate and acts as both an activator and a repressor. By genomic systematic evolution of ligands by exponential enrichment (SELEX) screening and promoter reporter assays, we have identified a total of nine operons, including the hitherto identified
modA
,
moaA
,
dmsA
, and
napF
operons, of which six were activated by ModE and three were repressed. In addition, two promoters were newly identified and direct transcription of novel genes, referred to as
morA
and
morB
, located on antisense strands of
yghW
and
torY
, respectively. The
morA
gene encodes a short peptide, MorA, with an unusual initiation codon. Surprisingly, overexpression of the
morA
5′ untranslated region exhibited an inhibitory influence on colony formation of
E. coli
K-12.
Pilin and Sortase Residues Critical for Endocarditis- and Biofilm-Associated Pilus Biogenesis in Enterococcus faecalis
Authors: Nielsen Hailyn V. , Flores-Mireles Ana L. , Kau Andrew L. , Kline Kimberly A. , Pinkner Jerome S. , Neiers Fabrice , Normark Staffan , Henriques-Normark Birgitta , Caparon Michael G. , Hultgren Scott J.
Published: August 2013

Enterococci commonly cause hospital-acquired infections, such as infective endocarditis and catheter-associated urinary tract infections. In animal models of these infections, a long hairlike extracellular protein fiber known as the endocarditis- and biofilm-associated (Ebp) pilus is an important virulence factor for
Enterococcus faecalis
. For Ebp and other sortase-assembled pili, the pilus-associated sortases are essential for fiber formation as they create covalent isopeptide bonds between the sortase recognition motif and the pilin-like motif of the pilus subunits. However, the molecular requirements governing the incorporation of the three pilus subunits (EbpA, EbpB, and EbpC) have not been investigated in
E. faecalis
. Here, we show that a Lys residue within the pilin-like motif of the EbpC subunit was necessary for EbpC polymerization. However, incorporation of EbpA into the pilus fiber only required its sortase recognition motif (LPXTG), while incorporation of EbpB only required its pilin-like motif. Only the sortase recognition motif would be required for incorporation of the pilus tip subunit, while incorporation of the base subunit would only require the pilin recognition motif. Thus, these data support a model with EbpA at the tip and EbpB at the base of an EbpC polymer. In addition, the housekeeping sortase, SrtA, was found to process EbpB and its predicted catalytic Cys residue was required for efficient cell wall anchoring of mature Ebp pili. Thus, we have defined molecular interactions involved in fiber polymerization, minor subunit organization, and pilus subcellular compartmentalization in the
E. faecalis
Ebp pilus system. These studies advance our understanding of unique molecular mechanisms of sortase-assembled pilus biogenesis.
Suppressor Analysis Reveals a Role for SecY in the SecA2-Dependent Protein Export Pathway of Mycobacteria
Authors: Ligon Lauren S. , Rigel Nathan W. , Romanchuk Artur , Jones Corbin D. , Braunstein Miriam
Published: August 2013

All bacteria use the conserved Sec pathway to transport proteins across the cytoplasmic membrane, with the SecA ATPase playing a central role in the process. Mycobacteria are part of a small group of bacteria that have two SecA proteins: the canonical SecA (SecA1) and a second, specialized SecA (SecA2). The SecA2-dependent pathway exports a small subset of proteins and is required for
Mycobacterium tuberculosis
virulence. The mechanism by which SecA2 drives export of proteins across the cytoplasmic membrane remains poorly understood. Here we performed suppressor analysis on a dominant negative
secA2
mutant (
secA2 K129R
) of the model mycobacterium
Mycobacterium smegmatis
to better understand the pathway used by SecA2 to export proteins. Two extragenic suppressor mutations were identified as mapping to the promoter region of
secY
, which encodes the central component of the canonical Sec export channel. These suppressor mutations increased
secY
expression, and this effect was sufficient to alleviate the
secA2 K129R
phenotype. We also discovered that the level of SecY protein was greatly diminished in the
secA2 K129R
mutant, but at least partially restored in the suppressors. Furthermore, the level of SecY in a suppressor strongly correlated with the degree of suppression. Our findings reveal a detrimental effect of SecA2 K129R on SecY, arguing for an integrated system in which SecA2 works with SecY and the canonical Sec translocase to export proteins.
How Escherichia coli Tolerates Profuse Hydrogen Peroxide Formation by a Catabolic Pathway
Authors: Ravindra Kumar Sripriya , Imlay James A.
Published: August 2013

When
Escherichia coli
grows on conventional substrates, it continuously generates 10 to 15 μM/s intracellular H
2
O
2
through the accidental autoxidation of redox enzymes. Dosimetric analyses indicate that scavenging enzymes barely keep this H
2
O
2
below toxic levels. Therefore, it seemed potentially problematic that
E. coli
can synthesize a catabolic phenylethylamine oxidase that stoichiometrically generates H
2
O
2
. This study was undertaken to understand how
E. coli
tolerates the oxidative stress that must ensue. Measurements indicated that phenylethylamine-fed cells generate H
2
O
2
at 30 times the rate of glucose-fed cells. Two tolerance mechanisms were identified. First, in enclosed laboratory cultures, growth on phenylethylamine triggered induction of the OxyR H
2
O
2
stress response. Null mutants (
ΔoxyR
) that could not induce that response were unable to grow. This is the first demonstration that OxyR plays a role in protecting cells against endogenous H
2
O
2
. The critical element of the OxyR response was the induction of H
2
O
2
scavenging enzymes, since mutants that lacked NADH peroxidase (Ahp) grew poorly, and those that additionally lacked catalase did not grow at all. Other OxyR-controlled genes were expendable. Second, phenylethylamine oxidase is an unusual catabolic enzyme in that it is localized in the periplasm. Calculations showed that when cells grow in an open environment, virtually all of the oxidase-generated H
2
O
2
will diffuse across the outer membrane and be lost to the external world, rather than enter the cytoplasm where H
2
O
2
-sensitive enzymes are located. In this respect, the periplasmic compartmentalization of phenylethylamine oxidase serves the same purpose as the peroxisomal compartmentalization of oxidases in eukaryotic cells.
Topoisomerase I (TopA) Is Recruited to ParB Complexes and Is Required for Proper Chromosome Organization during Streptomyces coelicolor Sporulation
Authors: Szafran Marcin , Skut Patrycja , Ditkowski Bartosz , Ginda Katarzyna , Chandra Govind , Zakrzewska-Czerwińska Jolanta , Jakimowicz Dagmara
Published: August 2013

Streptomycesspecies are bacteria that resemble filamentous fungi in their hyphal mode of growth and sporulation. InStreptomyces coelicolor, the conversion of multigenomic aerial hyphae into chains of unigenomic spores requires synchronized septation accompanied by segregation of tens of chromosomes into prespore compartments. The chromosome segregation is dependent on ParB protein, which assembles into an array of nucleoprotein complexes in the aerial hyphae. Here, we report that nucleoprotein ParB complexes are boundin vitroandin vivoby topoisomerase I, TopA, which is the only topoisomerase I homolog found inS. coelicolor. TopA cannot be eliminated, and its depletion inhibits growth and blocks sporulation. Surprisingly, sporulation in the TopA-depleted strain could be partially restored by deletion ofparB. Furthermore, the formation of regularly spaced ParB complexes, which is a prerequisite for proper chromosome segregation and septation during the development of aerial hyphae, has been found to depend on TopA. We hypothesize that TopA is recruited to ParB complexes during sporulation, and its activity is required to resolve segregating chromosomes.
TraG Encoded by the pIP501 Type IV Secretion System Is a Two-Domain Peptidoglycan-Degrading Enzyme Essential for Conjugative Transfer
Authors: Arends Karsten , Celik Ertugrul-Kaan , Probst Ines , Goessweiner-Mohr Nikolaus , Fercher Christian , Grumet Lukas , Soellue Cem , Abajy Mohammad Yaser , Sakinc Tuerkan , Broszat Melanie , Schiwon Katarzyna , Koraimann Guenther , Keller Walter , Grohmann Elisabeth
Published: August 2013

pIP501 is a conjugative broad-host-range plasmid frequently present in nosocomial
Enterococcus faecalis
and
Enterococcus faecium
isolates. We focus here on the functional analysis of the type IV secretion gene
traG
, which was found to be essential for pIP501 conjugative transfer between Gram-positive bacteria. The TraG protein, which localizes to the cell envelope of
E. faecalis
harboring pIP501, was expressed and purified without its N-terminal transmembrane helix (TraGΔTMH) and shown to possess peptidoglycan-degrading activity. TraGΔTMH was inhibited by specific lytic transglycosylase inhibitors hexa-
N
-acetylchitohexaose and bulgecin A. Analysis of the TraG sequence suggested the presence of two domains which both could contribute to the observed cell wall-degrading activity: an N-terminal soluble lytic transglycosylase domain (SLT) and a C-terminal cysteine-, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The protein domains were expressed separately, and both degraded peptidoglycan. A change of the conserved glutamate residue in the putative catalytic center of the SLT domain (E87) to glycine resulted in almost complete inactivity, which is consistent with this part of TraG being a predicted lytic transglycosylase. Based on our findings, we propose that TraG locally opens the peptidoglycan to facilitate insertion of the Gram-positive bacterial type IV secretion machinery into the cell envelope.