bluuebunny/crossref_metadata_2025 huggingface dataset:
Genomic insights into the fate of colistin resistance and Acinetobacter baumannii during patient treatment
Authors: Snitkin Evan S. , Zelazny Adrian M. , Gupta Jyoti ,   NISC Comparative Sequencing Program, Palmore Tara N. , Murray Patrick R. , Segre Julia A.
Published: April 2013

Bacterial whole-genome sequencing (WGS) of human pathogens has provided unprecedented insights into the evolution of antibiotic resistance. Most studies have focused on identification of resistance mutations, leaving one to speculate on the fate of these mutants once the antibiotic selective pressure is removed. We performed WGS on longitudinal isolates of Acinetobacter baumannii from patients undergoing colistin treatment, and upon subsequent drug withdrawal. In each of the four patients, colistin resistance evolved via mutations at the pmr locus. Upon colistin withdrawal, an ancestral susceptible strain outcompeted resistant isolates in three of the four cases. In the final case, resistance was also lost, but by a compensatory inactivating mutation in the transcriptional regulator of the pmr locus. Notably, this inactivating mutation reduced the probability of reacquiring colistin resistance when subsequently challenged in vitro. On face value, these results supported an in vivo fitness cost preventing the evolution of stable colistin resistance. However, more careful analysis of WGS data identified genomic evidence for stable colistin resistance undetected by clinical microbiological assays. Transcriptional studies validated this genomic hypothesis, showing increased pmr expression of the initial isolate. Moreover, altering the environmental growth conditions of the clinical assay recapitulated the classification as colistin resistant. Additional targeted sequencing revealed that this isolate evolved undetected in a patient undergoing colistin treatment, and was then transmitted to other hospitalized patients, further demonstrating its stability in the absence of colistin. This study provides a unique window into mutational pathways taken in response to antibiotic pressure in vivo, and demonstrates the potential for genome sequence data to predict resistance phenotypes.
The immature dentate gyrus represents a shared phenotype of mouse models of epilepsy and psychiatric disease
Authors: Shin Rick , Kobayashi Katsunori , Hagihara Hideo , Kogan Jeffrey H , Miyake Shinichi , Tajinda Katsunori , Walton Noah M , Gross Adam K , Heusner Carrie L , Chen Qian , Tamura Kouichi , Miyakawa Tsuyoshi , Matsumoto Mitsuyuki
Published: April 2013

Objectives

There is accumulating evidence to suggest psychiatric disorders, such as bipolar disorder and schizophrenia, share common etiologies, pathophysiologies, genetics, and drug responses with many of the epilepsies. Here, we explored overlaps in cellular/molecular, electrophysiological, and behavioral phenotypes between putative mouse models of bipolar disorder/schizophrenia and epilepsy. We tested the hypothesis that an immature dentate gyrus (iDG), whose association with psychosis in patients has recently been reported, represents a common phenotype of both diseases.

Methods

Behaviors of calcium/calmodulin‐dependent protein kinase II alpha (α‐CaMKII) heterozygous knock‐out (KO) mice, which are a representative bipolar disorder/schizophrenia model displaying iDG, and pilocarpine‐treated mice, which are a representative epilepsy model, were tested followed by quantitative polymerase chain reaction (qPCR)/immunohistochemistry for mRNA/protein expression associated with an iDG phenotype. In vitro electrophysiology of dentate gyrus granule cells (DG GCs) was examined in pilocarpine‐treated epileptic mice.

Results

The two disease models demonstrated similar behavioral deficits, such as hyperactivity, poor working memory performance, and social withdrawal. Significant reductions in mRNA expression and immunoreactivity of the mature neuronal marker calbindin and concomitant increases in mRNA expression and immunoreactivity of the immature neuronal marker calretinin represent iDG signatures that are present in both mice models. Electrophysiologically, we have confirmed that DG GCs from pilocarpine‐treated mice represent an immature state. A significant decrease in hippocampal α‐CaMKII protein levels was also found in both models.

Conclusions

Our data have shown iDG signatures from mouse models of both bipolar disorder/schizophrenia and epilepsy. The evidence suggests that the iDG may, in part, be responsible for the abnormal behavioral phenotype, and that the underlying pathophysiologies in epilepsy and bipolar disorder/schizophrenia are strikingly similar.
6q deletion detected by fluorescence in situ hybridization using bacterial artificial chromosome in chronic lymphocytic leukemia
Authors: Dalsass Alessia , Mestichelli Francesca , Ruggieri Miriana , Gaspari Paola , Pezzoni Valerio , Vagnoni Davide , Angelini Mario , Angelini Stefano , Bigazzi Catia , Falcioni Sadia , Troiani Emanuela , Alesiani Francesco , Catarini Massimo , Attolico Immacolata , Scortechini Ilaria , Discepoli Giancarlo , Galieni Piero
Published: April 2013

Deletions of the long arm of chromosome 6 are known to occur at relatively low frequency (3–6%) in chronic lymphocytic leukemia (CLL), and they are more frequently observed in 6q21. Few data have been reported regarding other bands on 6q involved by cytogenetic alterations in CLL. The cytogenetic study was performed in nuclei and metaphases obtained after stimulation with a combination of CpG‐oligonucleotide DSP30 and interleukin‐2. Four bacterial artificial chromosome (BAC) clones mapping regions in bands 6q16, 6q23, 6q25, 6q27 were used as probes for fluorescence in situ hybridization in 107 CLL cases in order to analyze the occurrence and localization of 6q aberrations. We identified 11 cases (10.2%) with 6q deletion of 107 patients studied with CLL. The trends of survival curves and the treatment‐free intervals (TFI) of patients with deletion suggest a better outcome than the other cytogenetic risk groups. We observed two subgroups with 6q deletion as the sole anomaly: two cases with 6q16 deletion, and three cases with 6q25.2–27 deletion. There were differences of age, stage, and TFI between both subgroups. By using BAC probes, we observed that 6q deletion has a higher frequency in CLL and is linked with a good prognosis. In addition, it was observed that the deletion in 6q16 appears to be the most frequent and, if present as the only abnormality, it could be associated with a most widespread disease.
Natural mortality estimators for information‐limited fisheries
Authors: Kenchington Trevor J
Published: April 2013

The 29 estimators of natural mortality (M) that have been proposed for ‘information‐limited’ fisheries are reviewed, together with a new alternative presented here. Each is applied to 13 example populations for which well‐founded estimates are available of bothMand the estimators' parameters. None of the 30 can provide accurate estimates for every species, and none appears sufficiently precise for use in analytical stock assessments, while several perform so poorly as to have no practical utility. If the growth coefficientKhas been reliably estimated, eitherM = 1.5 Kor Pauly's long‐established estimator can provide useful estimates ofM,but they fail with species that have long adult lives after swift juvenile growth, with those that never reach their asymptotic lengths and with species that otherwise deviate from archetypal teleost life histories. If a pre‐exploitation maximum observed age (Tmax) can be established,Mcan be estimated for both teleosts and sharks usingM = 4.3/Tmaxbut that seriously underestimates when the effective sample size (ne) is large and overestimates with species showing pronounced senescence. The new estimator presented here addressesnebut is upset by even mild senescence. Some estimators ofM‐at‐size, particularly ones recently advanced by Gislasonet al. and Charnovet al., also show promise but require further examination. It is recommended that fisheries scientists measureMby more advanced methods whenever possible. If ‘information‐limited’ estimators must be used, their uncertainties should be acknowledged and their errors propagated into management advice.
The geological significance of 40Ar/39Ar and Rb–Sr white mica ages from Syros and Sifnos, Greece: a record of continuous (re)crystallization during exhumation?
Authors: Bröcker M. , Baldwin S. , Arkudas R.
Published: April 2013

The Attic‐Cycladic crystalline belt in the central Aegean region records a complex structural and metamorphic evolution that documents Cenozoic subduction zone processes and exhumation. A prerequisite to develop an improved tectono‐metamorphic understanding of this area is dating of distinct P–T–D stages. To evaluate the geological significance of phengite ages of variably overprinted rocks, 40Ar/39Ar and Rb–Sr analyses were undertaken on transitional blueschist–greenschist and greenschist facies samples from the islands of Syros and Sifnos. White mica geochronology indicates a large age variability (40Ar/39Ar: 41–27 Ma; Rb–Sr: 34–20 Ma). Petrologically similar samples have either experienced greenschist facies overprinting at different times or variations in ages record variable degrees of greenschist facies retrogression and incomplete resetting of isotopic systematics. The 40Ar/39Ar and Rb–Sr data for metamorphic rocks from both islands record only minor, localized evidence for Miocene ages (c. 21 Ma) that are well documented elsewhere in the Cyclades and interpreted to result from retrogression of high‐pressure mineral assemblages during lower pressure metamorphism. Field and textural evidence suggests that heterogeneous overprinting may be due to a lack of permeability and/or limited availability of fluids in some bulk compositions and that retrogression was more or less parallel to lithological layering and/or foliation as a result of, possibly deformation‐enhanced, channelized fluid ingress. Published and new 40Ar/39Ar and Rb–Sr data for both islands indicate apparent age variations that can be broadly linked to mineral assemblages documenting transitional blueschist‐to‐greenschist‐ and/or greenschist facies metamorphism. The data do not record the timing of peak HP metamorphism, but may accurately record continuous (partial) resetting of isotopic systematics and/or (re)crystallization of white mica during exhumation and greenschist facies retrogression. The form of 40Ar/39Ar phengite age spectra are complex with the lowest temperature steps yielding Middle to Late Miocene ages. The youngest Rb–Sr ages suggest maximum ages of 20.6 ± 0.8 Ma (Syros) and 22.5 ± 0.6 Ma (Sifnos) for the timing of greenschist facies overprinting. The results of this study further accentuate the challenges of interpreting isotopic data for white mica from polymetamorphic terranes, particularly when mixing of populations and/or incomplete resetting of isotopic systematics occurs during exhumation. These data capture the full range of isotopic age variations in retrogressed HP rocks documented in previous isotopic studies, and can be interpreted in terms of the geodynamic evolution of the Aegean.
Differential expression of transcription factors Snail, Slug, SIP1, and Twist in ameloblastoma
Authors: Huat Siar Chong , Ng Kok Han
Published: April 2013

Background

Epithelial‐to‐mesenchymal transition (EMT) via the mechanism of transcription repression is a crucial process for the induction of invasiveness in many human tumors. Ameloblastoma is a benign odontogenic epithelial neoplasm with a locally infiltrative behavior. Twist, an EMT promoter, has been implicated in its invasiveness. The roles of the other transcription factors remain unclarified.

Materials and methods

Four transcription factors, namely Snail, Slug, SIP1, and Twist, were examined immunohistochemically in 64 ameloblastoma [18 unicystic (UA), 20 solid/multicystic (SA), 4 desmoplastic (DA), and 22 recurrent (RA)].

Results

All four transcription factors were differentially expressed in ameloblastoma [Snail: n = 60/64 (94%); Slug: n = 21/64 (33%); SIP: n = 18/64 (28%); Twist: n = 26/64 (41%)] (P < 0.05). Their distribution patterns were heterogeneous and were not significantly different between the tumor invasive front and central area (P > 0.05). Intracellular protein localization was predominantly nuclear for Snail, cytoplasmic>nuclear for Slug and SIP1, and cytoplasmic/nuclear for Twist. Overexpression of Snail in most subsets (UA = 18/18; SMA = 19/20; DA = 4/4; RA = 19/22) compared with the other transcription factors (P < 0.05) and selective expression for Slug, SIP1, and Twist in squamous/keratinous foci and at sites of epithelial cystic degeneration were among the main observations made. Stromal cells surrounding immunoreactive tumor cells tended to stain positive.

Conclusions

Present findings suggest that these transcription factors probably play differential roles in mediating local invasiveness in ameloblastoma. Overexpression of Snail in most subsets suggests that this molecule is most likely the prototype transcription factor involved in inducing EMT in the ameloblastoma.
Extremes of Nonlinear Vibration: Comparing Models Based on Moments, L-Moments, and Maximum Entropy
Authors: Winterstein Steven R. , MacKenzie Cameron A.
Published: April 2013

Wind and wave loads on offshore structures show nonlinear effects, which require non-Gaussian statistical models. Here we critically review the behavior of various non-Gaussian models. We first survey moment-based models; in particular, the four-moment “Hermite” model, a cubic transformation often used in wind and wave applications. We then derive an “L-Hermite” model, an alternative cubic transformation calibrated by the response “L-moments” rather than its ordinary statistical moments. These L-moments have recently found increasing use, in part because they show less sensitivity to distribution tails than ordinary moments. We find here, however, that these L-moments may not convey sufficient information to accurately estimate extreme response statistics. Finally, we show that four-moment maximum entropy models, also applied in the literature, may be inappropriate to model broader-than-Gaussian cases (e.g., responses to wind and wave loads).
Ultimate Compressive Capacity of Rectangular Plates With Partial Depth Pits
Authors: Jiang Xiaoli , Guedes Soares C.
Published: April 2013

Pitting corrosion has been one of the main corrosion types of immersed ship hulls, which can bring heavy damage and even accidents to in-service ships, particularly to aged ships. The aim of the present paper was to investigate the effects of pits on the ultimate compressive strength of mild steel plates under uniaxial compression. A series of nonlinear FEM analyses on plates with partial depth corrosion pits were carried out, changing the size, intensity and location of pits and the slenderness of plates. It was shown that the eccentricity induced by single side distributed pits had considerable degrading effects on the ultimate compressive capacity of plates. Although the degree of pit corrosion (DOP) did reflect the effect of pits to a large extent, it was not enough to rely on DOP exclusively to represent the extent of damage caused by pits, as “volume effect” should be considered.
Latching Control of an Oscillating Water Column Spar-Buoy Wave Energy Converter in Regular Waves
Authors: Henriques J. C. C. , Falcão A. F. O. , Gomes R. P. F. , Gato L. M. C.
Published: April 2013

The present paper concerns an oscillating water column (OWC) spar-buoy, possibly the simplest concept for a floating OWC wave energy converter. It is an axisymmetric device (and so insensitive to wave direction) consisting basically of a (relatively long) submerged vertical tail tube open at both ends and fixed to a floater that moves essentially in heave. The length of the tube determines the resonance frequency of the inner water column. The oscillating motion of the internal free surface relative to the buoy, produced by the incident waves, makes the air flow through a turbine that drives an electrical generator. It is well known that the frequency response of point absorbers like the spar buoy is relatively narrow, which implies that their performance in irregular waves is relatively poor. Phase control has been proposed to improve this situation. The present paper presents a theoretical investigation of phase control through the latching of an OWC spar-buoy in which the compressibility of air in the chamber plays an important role (the latching is performed by fast closing and opening an air valve in series with the turbine). In particular, such compressibility may remove the constraint of the latching threshold having to coincide with an instant of zero relative velocity between the two bodies (in the case under consideration, between the floater and the OWC). The modeling is performed in the time domain for a given device geometry and includes the numerical optimization of the air turbine rotational speed, chamber volume, and latching parameters. Results are obtained for regular waves.
Notes on the Prediction of Extreme Ship Response
Authors: Mao Wengang , Rychlik Igor
Published: April 2013

In this note the relation between two simple approaches to estimate the extreme ship response used when no, or a limited, amount of data are available is discussed. The first one employs the long term distribution of the local maxima of ship response while the second one uses the expected number of upcrossings of a level by the response. It is mathematically demonstrated that the two approaches are equivalent. However, the upcrossing method is more straightforward and convenient for practical applications, particularly for non-Gaussian responses. The full-scale measurements of a 2800 TEU container ship during the first six months of 2008 are used in the comparisons.
Identification and Characterization of a Tetramethylpyrazine Catabolic Pathway in Rhodococcus jostii TMP1
Authors: Kutanovas Simonas , Stankeviciute Jonita , Urbelis Gintaras , Tauraite Daiva , Rutkiene Rasa , Meskys Rolandas
Published: April 2013

At present, there are no published data on catabolic pathways of
N
-heterocyclic compounds, in which all carbon atoms carry a substituent. We identified the genetic locus and characterized key reactions in the aerobic degradation of tetramethylpyrazine in
Rhodococcus jostii
strain TMP1. By comparing protein expression profiles, we identified a tetramethylpyrazine-inducible protein of 40 kDa and determined its identity by tandem mass spectrometry (MS-MS)
de novo
sequencing. Searches against an
R. jostii
TMP1 genome database allowed the identification of the tetramethylpyrazine-inducible protein-coding gene. The tetramethylpyrazine-inducible gene was located within a 13-kb genome cluster, denominated the tetramethylpyrazine degradation (
tpd
) locus, that encoded eight proteins involved in tetramethylpyrazine catabolism. The genes from this cluster were cloned and transferred into tetramethylpyrazine-nondegrading
Rhodococcus erythropolis
strain SQ1. This allowed us to verify the function of the
tpd
locus, to isolate intermediate metabolites, and to reconstruct the catabolic pathway of tetramethylpyrazine. We report that the degradation of tetramethylpyrazine is a multistep process that includes initial oxidative aromatic-ring cleavage by tetramethylpyrazine oxygenase, TpdAB; subsequent hydrolysis by (
Z
)-
N
,
N
′-(but-2-ene-2,3-diyl)diacetamide hydrolase, TpdC; and further intermediate metabolite reduction by aminoalcohol dehydrogenase, TpdE. Thus, the genes responsible for bacterial degradation of pyrazines have been identified, and intermediate metabolites of tetramethylpyrazine degradation have been isolated for the first time.
Classification of Lytic Bacteriophages Attacking Dairy Leuconostoc Starter Strains
Authors: Ali Yahya , Kot Witold , Atamer Zeynep , Hinrichs Jï¿½rg , Vogensen Finn K. , Heller Knut J. , Neve Horst
Published: April 2013

A set of 83 lytic dairy bacteriophages (phages) infecting flavor-producing mesophilic starter strains of the
Leuconostoc
genus was characterized, and the first in-depth taxonomic scheme was established for this phage group. Phages were obtained from different sources, i.e., from dairy samples originating from 11 German dairies (50
Leuconostoc pseudomesenteroides
[
Ln. pseudomesenteroides
] phages, 4
Ln. mesenteroides
phages) and from 3 external phage collections (17
Ln. pseudomesenteroides
phages, 12
Ln. mesenteroides
phages). All phages belonged to the
Siphoviridae
family of phages with isometric heads (diameter, 55 nm) and noncontractile tails (length, 140 nm). With the exception of one phage (i.e., phage ΦLN25), all
Ln. mesenteroides
phages lysed the same host strains and revealed characteristic globular baseplate appendages. Phage ΦLN25, with different Y-shaped appendages, had a unique host range. Apart from two phages (i.e., phages P792 and P793), all
Ln. pseudomesenteroides
phages shared the same host range and had plain baseplates without distinguishable appendages. They were further characterized by the presence or absence of a collar below the phage head or by unique tails with straight striations. Phages P792 and P793 with characteristic fluffy baseplate appendages could propagate only on other specific hosts. All
Ln. mesenteroides
and all
Ln. pseudomesenteroides
phages were members of two (host species-specific) distinct genotypes but shared a limited conserved DNA region specifying their structural genes. A PCR detection system was established and was shown to be reliable for the detection of all
Leuconostoc
phage types.
Plasmid-Borne Type E Neurotoxin Gene Clusters in Clostridium botulinum Strains
Authors: Zhang Zhen , Hintsa Hannamari , Chen Ying , Korkeala Hannu , Lindstrï¿½m Miia
Published: April 2013

A collection of 36
Clostridium botulinum
type E strains was examined by pulsed-field gel electrophoresis (PFGE) and Southern hybridization with probes targeted to
botE
and
orfX1
in the neurotoxin gene cluster. Three strains were found to contain neurotoxin subtype E1 gene clusters in large plasmids of about 146 kb in size.
Defining the Functional Potential and Active Community Members of a Sediment Microbial Community in a High-Arctic Hypersaline Subzero Spring
Authors: Lay Chih-Ying , Mykytczuk Nadia C. S. , Yergeau ï¿½tienne , Lamarche-Gagnon Guillaume , Greer Charles W. , Whyte Lyle G.
Published: April 2013

The Lost Hammer (LH) Spring is the coldest and saltiest terrestrial spring discovered to date and is characterized by perennial discharges at subzero temperatures (−5ï¿½C), hypersalinity (salinity, 24%), and reducing (≈−165 mV), microoxic, and oligotrophic conditions. It is rich in sulfates (10.0%, wt/wt), dissolved H2S/sulfides (up to 25 ppm), ammonia (≈381 μM), and methane (11.1 g day−1). To determine its total functional and genetic potential and to identify its active microbial components, we performed metagenomic analyses of the LH Spring outlet microbial community and pyrosequencing analyses of the cDNA of its 16S rRNA genes. Reads related toCyanobacteria(19.7%),Bacteroidetes(13.3%), andProteobacteria(6.6%) represented the dominant phyla identified among the classified sequences. Reconstruction of the enzyme pathways responsible for bacterial nitrification/denitrification/ammonification and sulfate reduction appeared nearly complete in the metagenomic data set. In the cDNA profile of the LH Spring active community, ammonia oxidizers (Thaumarchaeota), denitrifiers (Pseudomonasspp.), sulfate reducers (Desulfobulbusspp.), and other sulfur oxidizers (Thermoprotei) were present, highlighting their involvement in nitrogen and sulfur cycling. Stress response genes for adapting to cold, osmotic stress, and oxidative stress were also abundant in the metagenome. Comparison of the composition of the functional community of the LH Spring to metagenomes from other saline/subzero environments revealed a close association between the LH Spring and another Canadian high-Arctic permafrost environment, particularly in genes related to sulfur metabolism and dormancy. Overall, this study provides insights into the metabolic potential and the active microbial populations that exist in this hypersaline cryoenvironment and contributes to our understanding of microbial ecology in extreme environments.
CmPEX6
, a Gene Involved in Peroxisome Biogenesis, Is Essential for Parasitism and Conidiation by the Sclerotial Parasite Coniothyrium minitans
Authors: Wei Wei , Zhu Wenjun , Cheng Jiasen , Xie Jiatao , Li Bo , Jiang Daohong , Li Guoqing , Yi Xianhong , Fu Yanping
Published: April 2013

Coniothyrium minitans
is a sclerotial parasite of the plant-pathogenic fungus
Sclerotinia sclerotiorum
, and conidial production and parasitism are two important aspects for commercialization of this biological control agent. To understand the mechanism of conidiation and parasitism at the molecular level, we constructed a transfer DNA (tDNA) insertional library with the wild-type strain ZS-1. A conidiation-deficient mutant, ZS-1TN22803, was uncovered through screening of this library. This mutant could produce pycnidia on potato dextrose agar (PDA), but most were immature and did not bear conidia. Moreover, this mutant lost the ability to parasitize or rot the sclerotia of
S. sclerotiorum
. Analysis of the tDNA flanking sequences revealed that a peroxisome biogenesis factor 6 (
PEX6
) homolog of
Saccharomyces cerevisiae
, named
CmPEX6
, was disrupted by the tDNA insertion in this mutant. Targeted gene replacement and gene complementation tests confirmed that a null mutation of
CmPEX6
was responsible for the phenotype of ZS-1TN22803. Further analysis showed that both ZS-1TN22803 and the targeted replacement mutants could not grow on PDA medium containing oleic acid, and they produced much less nitric oxide (NO) and hydrogen peroxide (H
2
O
2
) than wild-type strain ZS-1. The conidiation of ZS-1TN22803 was partially restored by adding acetyl-CoA or glyoxylic acid to the growth media. Our results suggest that fatty acid β-oxidation, reactive oxygen and nitrogen species, and possibly other unknown pathways in peroxisomes are involved in conidiation and parasitism by
C. minitans
.
Rational Design of a Novel Propeptide for Improving Active Production of Streptomyces griseus Trypsin in Pichia pastoris
Authors: Ling Zhenmin , Liu Yi , Teng Shaolei , Kang Zhen , Zhang Jingjing , Chen Jian , Du Guocheng
Published: April 2013

Applying
in silico
simulations and
in vitro
experiments, the amino acid proline was proved to have a profound influence on
Streptomyces griseus
trypsinogen, and the hydrogen bond between H
57
and D
102
was found to be crucial for trypsin activity. By introducing an artificial propeptide, IVEF, the titer of trypsin was increased 6.71-fold.
Potentiation of Mycovirus Transmission by Zinc Compounds via Attenuation of Heterogenic Incompatibility in Rosellinia necatrix
Authors: Ikeda Kenichi , Inoue Kanako , Kida Chiaki , Uwamori Takahiro , Sasaki Atsuko , Kanematsu Satoko , Park Pyoyun
Published: April 2013

Heterogenic incompatibility is considered a defense mechanism against deleterious intruders such as mycovirus.
Rosellinia necatrix
shows strong heterogenic incompatibility. In the heterogenic incompatibility reaction, the approaching hyphae hardly anastomosed, a distinctive barrage line formed, and green fluorescent protein (GFP)-labeled hyphae quickly lost their fluorescence when encountering incompatible hyphae. In this study, transmission of a hypovirulence-conferring mycovirus to strains with different genetic backgrounds was attempted. Various chemical reagents considered to affect the programmed cell death pathway or cell wall modification were examined. Treatment with zinc compounds was shown to aid in transmission of mycoviruses to strains with different genetic backgrounds. In incompatible pairings, treatment with zinc compounds accelerated hyphal anastomosis; moreover, cytosolic GFP was transmitted to the newly joined hyphae. These results suggest that zinc compounds not only increase hyphal anastomosis but also attenuate heterogenic incompatibility.
Roles of Alkyl Hydroperoxide Reductase Subunit C (AhpC) in Viable but Nonculturable Vibrio parahaemolyticus
Authors: Wang Hen-Wei , Chung Chun-Hui , Ma Tsung-Yong , Wong Hin-chung
Published: April 2013

Alkyl hydroperoxide reductase subunit C (AhpC) is the catalytic subunit responsible for the detoxification of reactive oxygen species that form in bacterial cells or are derived from the host; thus, AhpC facilitates the survival of pathogenic bacteria under environmental stresses or during infection. This study investigates the role of AhpC in the induction and maintenance of a viable but nonculturable (VBNC) state in
Vibrio parahaemolyticus
. In this investigation,
ahpC1
(VPA1683) and
ahpC2
(VP0580) were identified in chromosomes II and I of this pathogen, respectively. Mutants with deletions of these two
ahpC
genes and their complementary strains were constructed from the parent strain KX-V231. The growth of these strains was monitored on tryptic soy agar–3% NaCl in the presence of the extrinsic peroxides H
2
O
2
and
tert
-butyl hydroperoxide (
t
-BOOH) at different incubation temperatures. The results revealed that both
ahpC
genes were protective against
t
-BOOH, while
ahpC1
was protective against H
2
O
2
. The protective function of
ahpC2
at 4ï¿½C was higher than that of
ahpC1
. The times required to induce the VBNC state (4.7 weeks) at 4ï¿½C in a modified Morita mineral salt solution with 0.5% NaCl and then to maintain the VBNC state (4.7 weeks) in an
ahpC2
mutant and an
ahpC1 ahpC2
double mutant were significantly shorter than those for the parent strain (for induction, 6.2 weeks; for maintenance, 7.8 weeks) and the
ahpC1
mutant (for induction, 6.0 weeks; for maintenance, 8.0 weeks) (
P
< 0.03). Complementation with an
ahpC2
gene reversed the effects of the
ahpC2
mutation in shortening the times for induction and maintenance of the VBNC state. This investigation identified the different functions of the two
ahpC
genes and confirmed the particular role of
ahpC2
in the VBNC state of
V. parahaemolyticus
.
Imidazoacridinone Derivatives as Efficient Sensitizers in Photoantimicrobial Chemotherapy
Authors: Taraszkiewicz Aleksandra , Grinholc Mariusz , Bielawski Krzysztof P. , Kawiak Anna , Nakonieczna Joanna
Published: April 2013

The objective of this study was to investigate a new potential photosensitizer (PS) in the photodynamic inactivation (PDI) of microorganisms
in vitro
(11 reference strains and 13 clinical isolates, representing common Gram-positive and Gram-negative human pathogens), with special emphasis on
Candida albicans
. We studied the light-induced cytotoxicity of the imidazoacridinone derivative C1330 toward fungal cells grown in planktonic form. We examined the influence of various parameters (time of incubation, PDI quencher effect, and C1330 accumulation in
C. albicans
cells) on the efficacy of light-dependent cytotoxicity. Additionally, we checked for the potential cyto- and phototoxic activity of C1330 against human dermal keratinocytes. In our research, we used a broadband incoherent blue light source (380 to 470 nm) with an output power of 100 mW/cm
2
.
In vitro
studies showed that the C1330 action against
C. albicans
was a light-dependent process. C1330 was an efficient photosensitizer in the photodynamic inactivation of
C. albicans
, which reduced the growth of planktonic cells by 6.1 log
10
units. Efficient accumulation of PS in the nucleus and vacuoles was observed after 30 min of incubation, which correlated with the highest photokilling efficacy. Significant changes in intracellular structure were observed upon illumination of C1330-incubated
C. albicans
cells. In the case of the human HaCaT cell line, approximately 40% of cells survived the treatment, which indicates the potential benefit of further study of the application of C1330 in photoantimicrobial chemotherapy. These data suggest that PDI may be a viable approach for the treatment of localized
C. albicans
infections.
Iron and Copper Act Synergistically To Delay Anaerobic Growth of Bacteria
Authors: Bird Lina J. , Coleman Maureen L. , Newman Dianne K.
Published: April 2013

Transition metals are known to cause toxic effects through their interaction with oxygen, but toxicity under anoxic conditions is poorly understood. Here we investigated the effects of iron (Fe) and copper (Cu) on the anaerobic growth and gene expression of the purple phototrophic bacterium
Rhodopseudomonas palustris
TIE-1. We found that Fe(II) and Cu(II) act synergistically to delay anaerobic growth at environmentally relevant metal concentrations. Cu(I) and Cu(II) had similar effects both alone and in the presence of ascorbate, a Cu(II) reductant, indicating that reduction of Cu(II) to Cu(I) by Fe(II) is not sufficient to explain the growth inhibition. Addition of Cu(II) increased the toxicity of Co(II) and Ni(II); in contrast, Ni(II) toxicity was diminished in the presence of Fe(II). The synergistic anaerobic toxicity of Fe(II) and Cu(II) was also observed for
Escherichia coli
MG1655,
Shewanella oneidensis
MR-1, and
Rhodobacter capsulatus
SB1003. Gene expression analyses for
R. palustris
identified three regulatory genes that respond to Cu(II) and not to Fe(II): homologs of
cueR
and
cusR
, two known proteobacterial copper homeostasis regulators, and
csoR
, a copper regulator recently identified in
Mycobacterium tuberculosis
. Two P-type ATPase efflux pumps, along with an F
o
F
1
ATP synthase, were also upregulated by Cu(II) but not by Fe(II). An
Escherichia coli
mutant deficient in
copA
,
cus
, and
cueO
showed a smaller synergistic effect, indicating that iron might interfere with one or more of the copper homeostasis systems. Our results suggest that interactive effects of transition metals on microbial physiology may be widespread under anoxic conditions, although the molecular mechanisms remain to be more fully elucidated.
Identification of Genes Upregulated by the Transcription Factor Bcr1 That Are Involved in Impermeability, Impenetrability, and Drug Resistance of Candida albicans
a/
α Biofilms
Authors: Srikantha Thyagarajan , Daniels Karla J. , Pujol Claude , Kim Elena , Soll David R.
Published: April 2013

Candida albicans
forms two types of biofilm, depending upon the configuration of the mating type locus. Although architecturally similar,
a/
α biofilms are impermeable, impenetrable, and drug resistant, whereas
a/a
and α/α biofilms lack these traits. The difference appears to be the result of an alternative matrix. Overexpression in
a/a
cells of
BCR1
, a master regulator of the
a/
α matrix, conferred impermeability, impenetrability, and drug resistance to
a/a
biofilms. Deletion of
BCR1
in
a/
α cells resulted in the loss of these
a/
α-specific biofilm traits. Using
BCR1
overexpression in
a/a
cells, we screened 107 genes of interest and identified 8 that were upregulated by Bcr1. When each was overexpressed in
a/a
biofilms, the three
a/
α traits were partially conferred, and when each was deleted in
a/
α cells, the traits were partially lost. Five of the eight genes have been implicated in iron homeostasis, and six encode proteins that are either in the wall or plasma membrane or secreted. All six possess sites for O-linked and N-linked glycosylation that, like glycosylphosphatidylinositol (GPI) anchors, can cross-link to the wall and matrix, suggesting that they may exert a structural role in conferring impermeability, impenetrability, and drug resistance, in addition to their physiological functions. The fact that in a screen of 107 genes, all 8 of the Bcr1-upregulated genes identified play a role in impermeability, impenetrability, and drug resistance suggests that the formation of the
a/
α matrix is highly complex and involves a larger number of genes than the initial ones identified here.
Pathway of Glycine Betaine Biosynthesis in Aspergillus fumigatus
Authors: Lambou Karine , Pennati Andrea , Valsecchi Isabel , Tada Rui , Sherman Stephen , Sato Hajime , Beau Remi , Gadda Giovanni , Latgé Jean-Paul
Published: April 2013

The choline oxidase (CHOA) and betaine aldehyde dehydrogenase (BADH) genes identified inAspergillus fumigatusare present as a cluster specific for fungal genomes. Biochemical and molecular analyses of this cluster showed that it has very specific biochemical and functional features that make it unique and different from its plant and bacterial homologs.A. fumigatusChoAp catalyzed the oxidation of choline to glycine betaine with betaine aldehyde as an intermediate and reduced molecular oxygen to hydrogen peroxide using FAD as a cofactor.A. fumigatusBadhp oxidized betaine aldehyde to glycine betaine with reduction of NAD+to NADH. Analysis of theAfchoAΔ::HPHandAfbadAΔ::HPHsingle mutants and theAfchoAΔAfbadAΔ::HPHdouble mutant showed thatAfChoAp is essential for the use of choline as the sole nitrogen, carbon, or carbon and nitrogen source during the germination process.AfChoAp andAfBadAp were localized in the cytosol of germinating conidia and mycelia but were absent from resting conidia. Characterization of the mutant phenotypes showed that glycine betaine inA. fumigatusfunctions exclusively as a metabolic intermediate in the catabolism of choline and not as a stress protectant. This study inA. fumigatusis the first molecular, cellular, and biochemical characterization of the glycine betaine biosynthetic pathway in the fungal kingdom.
A Combination of Glycerol and Manganese Promotes Biofilm Formation in Bacillus subtilis via Histidine Kinase KinD Signaling
Authors: Shemesh Moshe , Chai Yunrong
Published: April 2013

The spore-forming bacterium
Bacillus subtilis
forms matrix-enclosed biofilms in response to environmental cues that to date remain poorly defined. Biofilm formation depends on the synthesis of an extracellular matrix, which is indirectly regulated by the transcriptional regulator Spo0A. The activity of Spo0A depends on its phosphorylation state. The level of phosphorylated Spo0A (Spo0A∼P) is controlled by a network of kinases and phosphatases, which respond to environmental and physiological signals. In spite of significant progress in understanding biofilm development, the fundamental question of how cells sense the environmental cues that trigger biofilm formation has largely remained unaddressed. Here, we report that biofilm formation of
B. subtilis
in LB medium is triggered by a combination of glycerol and manganese (GM). Moreover, LB medium with GM significantly stimulates biofilm-associated sporulation and production of an undefined brown pigment. We further show that transcription of the major operons responsible for matrix production and biofilm formation is dramatically enhanced in response to GM. We also establish that KinD is a principal histidine kinase responsible for sensing the presence of GM exclusively by its extracellular CACHE domain. Finally, we show that GM has a similar biofilm-promoting effect in two related
Bacillus
species,
B. licheniformis
and
B. cereus
, indicating that the biofilm-promoting effect of GM is conserved in
Bacillus
species.
The Escherichia coli Cpx Envelope Stress Response Regulates Genes of Diverse Function That Impact Antibiotic Resistance and Membrane Integrity
Authors: Raivio Tracy L. , Leblanc Shannon K. D. , Price Nancy L.
Published: April 2013

The Cpx envelope stress response mediates adaptation to stresses that cause envelope protein misfolding. Adaptation is partly conferred through increased expression of protein folding and degradation factors. The Cpx response also plays a conserved role in the regulation of virulence determinant expression and impacts antibiotic resistance. We sought to identify adaptive mechanisms that may be involved in these important functions by characterizing changes in the transcriptome of two different
Escherichia coli
strains when the Cpx response is induced. We show that, while there is considerable strain- and condition-specific variability in the Cpx response, the regulon is enriched for proteins and functions that are inner membrane associated under all conditions. Genes that were changed by Cpx pathway induction under all conditions were involved in a number of cellular functions and included several intergenic regions, suggesting that posttranscriptional regulation is important during Cpx-mediated adaptation. Some Cpx-regulated genes are centrally involved in energetics and play a role in antibiotic resistance. We show that a number of small, uncharacterized envelope proteins are Cpx regulated and at least two of these affect phenotypes associated with membrane integrity. Altogether, our work suggests new mechanisms of Cpx-mediated envelope stress adaptation and antibiotic resistance.
Identification of a Hotdog Fold Thioesterase Involved in the Biosynthesis of Menaquinone in Escherichia coli
Authors: Chen Minjiao , Ma Xinyu , Chen Xiaolei , Jiang Ming , Song Haigang , Guo Zhihong
Published: April 2013

Escherichia coli
is used as a model organism for elucidation of menaquinone biosynthesis, for which a hydrolytic step from 1,4-dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) to 1,4-dihydroxy-2-naphthoate is still unaccounted for. Recently, a hotdog fold thioesterase has been shown to catalyze this conversion in phylloquinone biosynthesis, suggesting that its closest homolog, YbgC in
Escherichia coli
, may be the DHNA-CoA thioesterase in menaquinone biosynthesis. However, this possibility is excluded by the involvement of YbgC in the Tol-Pal system and its complete lack of hydrolytic activity toward DHNA-CoA. To identify the hydrolytic enzyme, we have performed an activity-based screen of all nine
Escherichia coli
hotdog fold thioesterases and found that YdiI possesses a high level of hydrolytic activity toward DHNA-CoA, with high substrate specificity, and that another thioesterase, EntH, from siderophore biosynthesis exhibits a moderate, much lower DHNA-CoA thioesterase activity. Deletion of the
ydiI
gene from the bacterial genome results in a significant decrease in menaquinone production, which is little affected in Δ
ybgC
and Δ
entH
mutants. These results support the notion that YdiI is the DHNA-CoA thioesterase involved in the biosynthesis of menaquinone in the model bacterium.
Crystal Structure and Site-Directed Mutagenesis Analyses of Haloalkane Dehalogenase LinB from Sphingobium sp. Strain MI1205
Authors: Okai Masahiko , Ohtsuka Jun , Imai Lica Fabiana , Mase Tomoko , Moriuchi Ryota , Tsuda Masataka , Nagata Koji , Nagata Yuji , Tanokura Masaru
Published: April 2013

The enzymes LinB
UT
and LinB
MI
(LinB from
Sphingobium japonicum
UT26 and
Sphingobium
sp. MI1205, respectively) catalyze the hydrolytic dechlorination of β-hexachlorocyclohexane (β-HCH) and yield different products, 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL), respectively, despite their 98% identity in amino acid sequence. To reveal the structural basis of their different enzymatic properties, we performed site-directed mutagenesis and X-ray crystallographic studies of LinB
MI
and its seven point mutants. The mutation analysis revealed that the seven amino acid residues uniquely found in LinB
MI
were categorized into three groups based on the efficiency of the first-step (from β-HCH to PCHL) and second-step (from PCHL to TCDL) conversions. Crystal structure analyses of wild-type LinB
MI
and its seven point mutants indicated how each mutated residue contributed to the first- and second-step conversions by LinB
MI
. The dynamics simulation analyses of wild-type LinB
MI
and LinB
UT
revealed that the entrance of the substrate access tunnel of LinB
UT
was more flexible than that of LinB
MI
, which could lead to the different efficiencies of dehalogenation activity between these dehalogenases.
Subdomains N2N3 of Fibronectin Binding Protein A Mediate Staphylococcus aureus Biofilm Formation and Adherence to Fibrinogen Using Distinct Mechanisms
Authors: Geoghegan Joan A. , Monk Ian R. , O'Gara James P. , Foster Timothy J.
Published: April 2013

Health care-associated methicillin-resistant
Staphylococcus aureus
(HA-MRSA) forms biofilm
in vitro
that is dependent on the surface-located fibronectin binding proteins A and B (FnBPA, FnBPB). Here we provide new insights into the requirements for FnBP-dependent biofilm formation by MRSA. We show that expression of FnBPs is sustained at high levels throughout the growth cycle in the HA-MRSA strain BH1CC in contrast to laboratory strain SH1000, where expression could be detected only in exponential phase. We found that FnBP-mediated biofilm accumulation required Zn
2+
, while the removal of Zn
2+
had no effect on the ability of FnBPA to mediate bacterial adherence to fibrinogen. We also investigated the role of FnBPA expressed on the surface of
S. aureus
in promoting biofilm formation and bacterial adhesion to fibrinogen. The minimum part of FnBPA required for ligand binding has so far been defined only with recombinant proteins. Here we found that the N1 subdomain was not required for biofilm formation or for FnBPA to promote bacterial adherence to fibrinogen. Residues at the C terminus of subdomain N3 required for FnBPA to bind to ligands using the “dock, lock, and latch” mechanism were necessary for FnBPA to promote bacterial adherence to fibrinogen. However, these residues were not necessary to form biofilm, allowing us to localize the region of FnBPA required for biofilm accumulation to residues 166 to 498. Thus, FnBPA mediates biofilm formation and bacterial adhesion to fibrinogen using two distinct mechanisms. Finally, we identified a hitherto-unrecognized thrombin cleavage site close to the boundary between subdomains N1 and N2 of FnBPA.
Utilization of
d
-Ribitol by Lactobacillus casei BL23 Requires a Mannose-Type Phosphotransferase System and Three Catabolic Enzymes
Authors: Bourand A. , Yebra M. J. , Boël G. , Mazé A. , Deutscher J.
Published: April 2013

Lactobacillus casei
strains 64H and BL23, but not ATCC 334, are able to ferment
d
-ribitol (also called
d
-adonitol). However, a BL23-derived
ptsI
mutant lacking enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was not able to utilize this pentitol, suggesting that strain BL23 transports and phosphorylates
d
-ribitol via a PTS. We identified an 11-kb region in the genome sequence of
L. casei
strain BL23 (
LCABL_29160
to
LCABL_29270
) which is absent from strain ATCC 334 and which contains the genes for a GlpR/IolR-like repressor, the four components of a mannose-type PTS, and six metabolic enzymes potentially involved in
d
-ribitol metabolism. Deletion of the gene encoding the EIIB component of the presumed ribitol PTS indeed prevented
d
-ribitol fermentation. In addition, we overexpressed the six catabolic genes, purified the encoded enzymes, and determined the activities of four of them. They encode a
d
-ribitol-5-phosphate (
d
-ribitol-5-P) 2-dehydrogenase, a
d
-ribulose-5-P 3-epimerase, a
d
-ribose-5-P isomerase, and a
d
-xylulose-5-P phosphoketolase. In the first catabolic step, the protein
d
-ribitol-5-P 2-dehydrogenase uses NAD
+
to oxidize
d
-ribitol-5-P formed during PTS-catalyzed transport to
d
-ribulose-5-P, which, in turn, is converted to
d
-xylulose-5-P by the enzyme
d
-ribulose-5-P 3-epimerase. Finally, the resulting
d
-xylulose-5-P is split by
d
-xylulose-5-P phosphoketolase in an inorganic phosphate-requiring reaction into acetylphosphate and the glycolytic intermediate
d
-glyceraldehyde-3-P. The three remaining enzymes, one of which was identified as
d
-ribose-5-P-isomerase, probably catalyze an alternative ribitol degradation pathway, which might be functional in
L. casei
strain 64H but not in BL23, because one of the BL23 genes carries a frameshift mutation.