bluuebunny/crossref_metadata_2025_split huggingface dataset:
Next-Generation Sequencing of Microbial Communities in the Athabasca River and Its Tributaries in Relation to Oil Sands Mining Activities
Authors: Yergeau Etienne , Lawrence John R. , Sanschagrin Sylvie , Waiser Marley J. , Korber Darren R. , Greer Charles W. 
Published: August 2012

ABSTRACT

The Athabasca oil sands deposit is the largest reservoir of crude bitumen in the world. Recently, the soaring demand for oil and the availability of modern bitumen extraction technology have heightened exploitation of this reservoir and the potential unintended consequences of pollution in the Athabasca River. The main objective of the present study was to evaluate the potential impacts of oil sands mining on neighboring aquatic microbial community structure. Microbial communities were sampled from sediments in the Athabasca River and its tributaries as well as in oil sands tailings ponds. Bacterial and archaeal 16S rRNA genes were amplified and sequenced using next-generation sequencing technology (454 and Ion Torrent). Sediments were also analyzed for a variety of chemical and physical characteristics. Microbial communities in the fine tailings of the tailings ponds were strikingly distinct from those in the Athabasca River and tributary sediments. Microbial communities in sediments taken close to tailings ponds were more similar to those in the fine tailings of the tailings ponds than to the ones from sediments further away. Additionally, bacterial diversity was significantly lower in tailings pond sediments. Several taxonomic groups of
Bacteria
and
Archaea
showed significant correlations with the concentrations of different contaminants, highlighting their potential as bioindicators. We also extensively validated Ion Torrent sequencing in the context of environmental studies by comparing Ion Torrent and 454 data sets and by analyzing control samples.
Unveiling the Expression Characteristics of IspC, a Cell Wall-Associated Peptidoglycan Hydrolase in Listeria monocytogenes, during Growth under Stress Conditions
Authors: Ronholm Jennifer , Cao Xudong , Lin Min 
Published: August 2012

ABSTRACT

Listeria monocytogenes
serotype 4b is a food-borne pathogen of public health concern, since it accounts for approximately 40% of human listeriosis cases. We have recently identified IspC, a surface-localized peptidoglycan hydrolase, as the antigen recognized by a number of monoclonal antibodies (MAbs) produced against a serotype 4b strain for diagnostic applications. To determine whether IspC, which is well conserved among various serotype 4b strains, is a useful diagnostic marker in antibody-based methods, we assessed the expression of IspC in
L. monocytogenes
cultured under normal and stress conditions. A functional promoter directing the transcription of the
ispC
gene was identified upstream of the
ispC
open reading frame by constructing a promoterless
lacZ
gene fusion with the putative
ispC
promoter region and by 5′ rapid amplification of cDNA ends analysis. Using both the
lacZ
reporter gene system and immunofluorescent staining with an IspC-specific MAb, we provide evidence that IspC is expressed on the cell surface in all growth conditions tested (temperature, osmotic stress, pH, ethanol, oxidative stress, anaerobic conditions, carbon source, and type of growth media) that allow for cellular division, although the level of
ispC
gene expression varies. These results demonstrated the usefulness of IspC as an excellent diagnostic marker for the serotype 4b strains and imply that IspC, in conjunction with specific MAbs, can be targeted for detection and isolation of
L. monocytogenes
serotype 4b strains directly from food, environmental, and clinical samples with minimal or no need for culture enrichment.
Molecular Characterization of Fluoroquinolone-Resistant Aeromonas spp. Isolated from Imported Shrimp
Authors: Shakir Zakiya , Khan Saeed , Sung Kidon , Khare Sangeeta , Khan Ashraf , Steele Roger , Nawaz Mohamed 
Published: August 2012

ABSTRACT

Sixty-three nalidixic acid-resistant
Aeromonas
sp. isolates were obtained from imported shrimp. Phylogenetic analysis of
gyrB
sequences indicated that 18 were
A. enteropelogenes
, 26 were
A. caviae
, and 19 were
A. sobria
. Double missense mutations in the quinolone resistance-determining region (QRDR) of
gyrA
at codon 83 (Ser→Val/Ile) and codon 92 (Leu→Met) coupled with a point mutation of
parC
at codon 80 (Ser→Ile/Phe) conferred high levels of quinolone resistance in the isolates. A majority of
A. enteropelogenes
and
A. caviae
strains harbored toxin genes, whereas only a few
A. sobria
strains harbored these genes. The fluoroquinolone-resistant
Aeromonas
spp. exhibited higher cytotoxicity than fluoroquinolone-sensitive, virulent
Aeromonas
spp. to rat epithelial cells.
Genomic and Transcriptomic Studies of an RDX (Hexahydro-1,3,5-Trinitro-1,3,5-Triazine)-Degrading Actinobacterium
Authors: Chen Hao-Ping , Zhu Song-Hua , Casabon Israël , Hallam Steven J. , Crocker Fiona H. , Mohn William W. , Indest Karl J. , Eltis Lindsay D. 
Published: August 2012

ABSTRACT

Whole-genome sequencing, transcriptomic analyses, and metabolic reconstruction were used to investigate
Gordonia
sp. strain KTR9's ability to catabolize a range of compounds, including explosives and steroids. Aspects of this mycolic acid-containing actinobacterium's catabolic potential were experimentally verified and compared with those of rhodococci and mycobacteria.
Iterative Combinatorial Mutagenesis as an Effective Strategy for Generation of Deacetoxycephalosporin C Synthase with Improved Activity toward Penicillin G
Authors: Ji Junjie , Fan Keqiang , Tian Xiuyun , Zhang Xia , Zhang Yuxiu , Yang Keqian 
Published: August 2012

ABSTRACT

An iterative combinatorial mutagenesis (ICM) strategy was used to engineer deacetoxycephalosporin C synthase of
Streptomyces clavuligerus
(scDAOCS) for improved activity toward penicillin G. Seven mutational sites were repeatedly combined onto a starter mutant (C155Y Y184H V275I C281Y) of scDAOCS. Eleven improved combinatorial mutants were identified from 24 mutants in four rounds of ICM.
Survival and Germination of Bacillus cereus Spores without Outgrowth or Enterotoxin Production during
In Vitro
Simulation of Gastrointestinal Transit
Authors: Ceuppens Siele , Uyttendaele Mieke , Drieskens Katrien , Heyndrickx Marc , Rajkovic Andreja , Boon Nico , Van de Wiele Tom 
Published: August 2012

ABSTRACT

To study the gastrointestinal survival and enterotoxin production of the food-borne pathogen
Bacillus cereus
, an
in vitro
simulation experiment was developed to mimic gastrointestinal passage in 5 phases: (i) the mouth, (ii) the stomach, with gradual pH decrease and fractional emptying, (iii) the duodenum, with high concentrations of bile and digestive enzymes, (iv) dialysis to ensure bile reabsorption, and (v) the ileum, with competing human intestinal bacteria. Four different
B. cereus
strains were cultivated and sporulated in mashed potato medium to obtain an inoculum of 7.0 log spores/ml. The spores showed survival and germination during the
in vitro
simulation of gastrointestinal passage, but vegetative outgrowth of the spores was suppressed by the intestinal bacteria during the final ileum phase. No bacterial proliferation or enterotoxin production was observed, despite the high inoculum levels. Little strain variability was observed: except for the psychrotrophic food isolate, the spores of all strains survived well throughout the gastrointestinal passage. The
in vitro
simulation experiments investigated the survival and enterotoxin production of
B. cereus
in the gastrointestinal lumen. The results obtained support the hypothesis that localized interaction of
B. cereus
with the host's epithelium is required for diarrheal food poisoning.
Versatility in Corrinoid Salvaging and Remodeling Pathways Supports Corrinoid-Dependent Metabolism in Dehalococcoides mccartyi
Authors: Yi Shan , Seth Erica C. , Men Yu-Jie , Stabler Sally P. , Allen Robert H. , Alvarez-Cohen Lisa , Taga Michiko E. 
Published: August 2012

ABSTRACTCorrinoids are cobalt-containing molecules that function as enzyme cofactors in a wide variety of organisms but are produced solely by a subset of prokaryotes. Specific corrinoids are identified by the structure of their axial ligands. The lower axial ligand of a corrinoid can be a benzimidazole, purine, or phenolic compound. Though it is known that many organisms obtain corrinoids from the environment, the variety of corrinoids that can serve as cofactors for any one organism is largely unstudied. Here, we examine the range of corrinoids that function as cofactors for corrinoid-dependent metabolism inDehalococcoides mccartyistrain 195.Dehalococcoidesbacteria play an important role in the bioremediation of chlorinated solvents in the environment because of their unique ability to convert the common groundwater contaminants perchloroethene and trichloroethene to the innocuous end product ethene. All isolatedD. mccartyistrains require exogenous corrinoids such as vitamin B12for growth. However, like many other corrinoid-dependent bacteria, none of the well-characterizedD. mccartyistrains has been shown to be capable of synthesizing corrinoidsde novo. In this study, we investigate the ability ofD. mccartyistrain 195 to use specific corrinoids, as well as its ability to modify imported corrinoids to a functional form. We show that strain 195 can use only specific corrinoids containing benzimidazole lower ligands but is capable of remodeling other corrinoids by lower ligand replacement when provided a functional benzimidazole base. This study of corrinoid utilization and modification byD. mccartyiprovides insight into the array of strategies that microorganisms employ in acquiring essential nutrients from the environment.
Heat Resistance and Salt Hypersensitivity in Lactococcus lactis Due to Spontaneous Mutation of
llmg_1816
(
gdpP
) Induced by High-Temperature Growth
Authors: Smith William M. , Pham Thi Huong , Lei Lin , Dou Junchao , Soomro Aijaz H. , Beatson Scott A. , Dykes Gary A. , Turner Mark S. 
Published: August 2012

ABSTRACT

During construction of several gene deletion mutants in
Lactococcus lactis
MG1363 which involved a high-temperature (37.5°C) incubation step, additional spontaneous mutations were observed which resulted in stable heat resistance and in some cases salt-hypersensitive phenotypes. Whole-genome sequencing of one strain which was both heat resistant and salt hypersensitive, followed by PCR and sequencing of four other mutants which shared these phenotypes, revealed independent mutations in
llmg_1816
in all cases. This gene encodes a membrane-bound stress signaling protein of the GdpP family, members of which exhibit cyclic dimeric AMP (c-di-AMP)-specific phosphodiesterase activity. Mutations were predicted to lead to single amino acid substitutions or protein truncations. An independent
llmg_1816
mutant (Δ1816), created using a suicide vector, also displayed heat resistance and salt hypersensitivity phenotypes which could be restored to wild-type levels following plasmid excision.
L. lactis
Δ1816 also displayed improved growth in response to sublethal concentrations of penicillin G. High-temperature incubation of a wild-type industrial
L. lactis
strain also resulted in spontaneous mutation of
llmg_1816
and heat-resistant and salt-hypersensitive phenotypes, suggesting that this is not a strain-specific phenomenon and that it is independent of a plasmid integration event. Acidification of milk by the
llmg_1816
-altered strain was inhibited by lower salt concentrations than the parent strain. This study demonstrates that spontaneous mutations can occur during high-temperature growth of
L. lactis
and that inactivation of
llmg_1816
leads to temperature resistance and salt hypersensitivity.
Functional and Physical Interactions among Saccharomyces cerevisiae α-Factor Receptors
Authors: Gehret Austin U. , Connelly Sara M. , Dumont Mark E. 
Published: August 2012

ABSTRACT

The α-factor receptor Ste2p is a G protein-coupled receptor (GPCR) expressed on the surface of
MAT
a
haploid cells of the yeast
Saccharomyces cerevisiae
. Binding of α-factor to Ste2p results in activation of a heterotrimeric G protein and of the pheromone response pathway. Functional interactions between α-factor receptors, such as dominant-negative effects and recessive behavior of constitutive and hypersensitive mutant receptors, have been reported previously. We show here that dominant-negative effects of mutant receptors persist over a wide range of ratios of the abundances of G protein to receptor and that such effects are not blocked by covalent fusion of G protein α subunits to normal receptors. In addition, we detected dominant effects of mutant C-terminally truncated receptors, which had not been previously reported to act in a dominant manner. Furthermore, coexpression of C-terminally truncated receptors with constitutively active mutant receptors results in enhancement of constitutive signaling. Together with previous evidence for oligomerization of Ste2p receptors, these results are consistent with the idea that functional interactions between coexpressed receptors arise from physical interactions between them rather than from competition for limiting downstream components, such as G proteins.
Gain-of-Function Mutations in
UPC2
Are a Frequent Cause of
ERG11
Upregulation in Azole-Resistant Clinical Isolates of Candida albicans
Authors: Flowers Stephanie A. , Barker Katherine S. , Berkow Elizabeth L. , Toner Geoffrey , Chadwick Sean G. , Gygax Scott E. , Morschhäuser Joachim , Rogers P. David 
Published: August 2012

ABSTRACT

In
Candida albicans
, Upc2 is a zinc-cluster transcription factor that targets genes, including those of the ergosterol biosynthesis pathway. To date, three documented
UPC2
gain-of-function (GOF) mutations have been recovered from fluconazole-resistant clinical isolates that contribute to an increase in
ERG11
expression and decreased fluconazole susceptibility. In a group of 63 isolates with reduced susceptibility to fluconazole, we found that 47 overexpressed
ERG11
by at least 2-fold over the average expression levels in 3 unrelated fluconazole-susceptible strains. Of those 47 isolates, 29 contained a mutation in
UPC2
, whereas the remaining 18 isolates did not. Among the isolates containing mutations in
UPC2
, we recovered eight distinct mutations resulting in putative single amino acid substitutions: G648D, G648S, A643T, A643V, Y642F, G304R, A646V, and W478C. Seven of these resulted in increased
ERG11
expression, increased cellular ergosterol, and decreased susceptibility to fluconazole compared to the results for the wild-type strain. Genome-wide transcriptional analysis was performed for the four strongest Upc2 amino acid substitutions (A643V, G648D, G648S, and Y642F). Genes commonly upregulated by all four mutations included those involved in ergosterol biosynthesis, in oxidoreductase activity, the major facilitator efflux pump encoded by the
MDR1
gene, and the uncharacterized ATP binding cassette transporter
CDR11
. These findings demonstrate that gain-of-function mutations in
UPC2
are more prevalent among clinical isolates than previously thought and make a significant contribution to azole antifungal resistance, but the findings do not account for
ERG11
overexpression in all such isolates of
C. albicans
.
The “Finger,” a Unique Multicellular Morphology of Candida albicans Induced by CO
2
and Dependent upon the Ras1-Cyclic AMP Pathway
Authors: Daniels Karla J. , Pujol Claude , Srikantha Thyagarajan , Soll David R. 
Published: August 2012

ABSTRACT

Most experiments exploring the basic biology of pathogenic microbes are performed
in vitro
under conditions that do not usually mimic those of their host niche. Hence, developmental programs initiated by specific host cues may be missed
in vitro
. We have tested the effects of growing low-density agar cultures of the yeast pathogen
Candida albicans
in concentrations of CO
2
found in the gastrointestinal tract. It is demonstrated that in physiological concentrations of CO
2
at 37°C, yeast cells form a heretofore undescribed multicellular “finger” morphology distinct from a previously described stalk-like structure induced by high doses of UV irradiation that kills more than 99.99% of cells. The finger extends aerially, is uniform in diameter, and is visible to the naked eye, attaining lengths of 3 mm. It is composed of a basal yeast cell monolayer adhering to a semispherical crater formed in the agar and connected to a basal bulb of yeast cells at a fragile interface. The bulb extends into the long shaft. We propose that a single, centrally located hypha extending the length of the shaft forms buds at compartment junctions that serve as the source of the yeast cells in the shaft. A mutational analysis reveals finger formation is dependent upon the pathway Ras1→Cdc35→cyclic AMP (cAMP) (PDE2—|)→Tpk2→Tec1. Because of the mechanically fragile interface and the compactness of bulb and shaft, we suggest that the finger may function as a multicellular dispersal mechanism produced in host niches containing high levels of CO
2
.
PAS Domain Residues and Prosthetic Group Involved in BdlA-Dependent Dispersion Response by Pseudomonas aeruginosa Biofilms
Authors: Petrova Olga E. , Sauer Karin 
Published: August 2012

ABSTRACT

Biofilm dispersion by
Pseudomonas aeruginosa
in response to environmental cues is dependent on the cytoplasmic BdlA protein harboring two sensory PAS domains and a chemoreceptor domain, TarH. The closest known and previously characterized BdlA homolog is the flavin adenine dinucleotide (FAD)-binding Aer, the redox potential sensor and aerotaxis transducer in
Escherichia coli
. Here, we made use of alanine replacement mutagenesis of the BdlA PAS domain residues previously demonstrated to be essential for aerotaxis in Aer to determine whether BdlA is a potential sensory protein. Five substitutions (D14A, N23A, W60A, I109A, and W182A) resulted in a null phenotype for dispersion. One protein, the BdlA protein with the G31A mutation (BdlA-G31A), transmitted a constant signal-on bias as it rendered
P. aeruginosa
biofilms hyperdispersive. The hyperdispersive phenotype correlated with increased interaction of BdlA-G31A with the phosphodiesterase DipA under biofilm growth conditions, resulting in increased phosphodiesterase activity and reduced biofilm biomass accumulation. We furthermore demonstrate that BdlA is a heme-binding protein. None of the BdlA protein variants analyzed led to a loss of the heme prosthetic group. The N-terminal PASa domain was identified as the heme-binding domain of BdlA, with BdlA-dependent nutrient-induced dispersion requiring the PASa domain. The findings suggest that BdlA plays a role in intracellular sensing of dispersion-inducing conditions and together with DipA forms a regulatory network that modulates an intracellular cyclic d-GMP (c-di-GMP) pool to enable dispersion.
Characterization of Axial and Proximal Histidine Mutations of the Decaheme Cytochrome MtrA from Shewanella sp. Strain ANA-3 and Implications for the Electron Transport System
Authors: Reyes Carolina , Qian Fang , Zhang Alissa , Bondarev Sergey , Welch Angel , Thelen Michael P. , Saltikov Chad W. 
Published: August 2012

ABSTRACTExtracellular respiration of solid-phase electron acceptors in some microorganisms requires a complex chain of multihemec-type cytochromes that span the inner and outer membranes. InShewanellaspecies, MtrA, an ∼35-kDa periplasmic decahemec-type cytochrome, is an essential component for extracellular respiration of iron(III). The exact mechanism of electron transport has not yet been resolved, but the arrangement of the polypeptide chain may have a strong influence on the capability of the MtrA cytochrome to transport electrons. The iron hemes of MtrA are bound to its polypeptide chain via proximal (CXXCH) and distal histidine residues. In this study, we show the effects of mutating histidine residues of MtrA to arginine on protein expression and extracellular respiration usingShewanellasp. strain ANA-3 as a model organism. Individual mutations to six out of nine proximal histidines in CXXCH of MtrA led to decreased protein expression. However, distal histidine mutations resulted in various degrees of protein expression. In addition, the effects of histidine mutations on extracellular respiration were tested using ferrihydrite and current production in microbial fuel cells. These results show that proximal histidine mutants were unable to reduce ferrihydrite. Mutations to the distal histidine residues resulted in various degrees of ferrihydrite reduction. These findings indicate that mutations to the proximal histidine residues affect MtrA expression, leading to loss of extracellular respiration ability. In contrast, mutations to the distal histidine residues are less detrimental to protein expression, and extracellular respiration can proceed.
Burkholderia xenovorans RcoM

Bx

-1, a Transcriptional Regulator System for Sensing Low and Persistent Levels of Carbon Monoxide
Authors: Kerby Robert L. , Roberts Gary P. 
Published: August 2012

ABSTRACT

The single-component RcoM transcription factor couples an N-terminally bound heme cofactor with a C-terminal “LytTR” DNA-binding domain. Here the RcoM

Bx

-1 protein from
Burkholderia xenovorans
LB400 was heterologously expressed and then purified in a form with minimal bound CO (∼10%) and was found to stably bind this effector with a nanomolar affinity. DNase I protection assays demonstrated that the CO-associated form binds with a micromolar affinity to two ∼60-bp DNA regions, each comprised of a novel set of three direct-repeat binding sites spaced 21 bp apart on center. Binding to each region was independent, while binding to the triplet binding sites within a region was cooperative, depended upon spacing and sequence, and was marked by phased DNase I hyperactivity and protection patterns consistent with considerable changes in the DNA conformation of the nucleoprotein complex. Each protected binding site spanned a conserved motif (5′-TTnnnG-3′) that was present, in triplicate, in putative RcoM-binding regions of more than a dozen organisms.
In vivo
screens confirmed the functional importance of the conserved “TTnnnG” motif residues and their triplet arrangement and were also used to determine an improved binding motif [5′-CnnC(C/A)(G/A)TTCAnG-3′] that more closely corresponds to canonical LytTR domain/DNA-binding sites. A low-affinity but CO-dependent binding of RcoM

Bx

-1 to a variety of DNA probes was demonstrated
in vitro
. We posit that for the RcoM

Bx

-1 protein, the high CO affinity combined with multiple low-affinity DNA-binding events constitutes a transcriptional “accumulating switch” that senses low but persistent CO levels.