bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection and Quantification of Functional Genes of Cellulose- Degrading, Fermentative, and Sulfate-Reducing Bacteria and Methanogenic Archaea
Authors: Pereyra L. P. , Hiibel S. R. , Prieto Riquelme M. V. , Reardon K. F. , Pruden A. 
Published: February 2010

ABSTRACT

Cellulose degradation, fermentation, sulfate reduction, and methanogenesis are microbial processes that coexist in a variety of natural and engineered anaerobic environments. Compared to the study of 16S rRNA genes, the study of the genes encoding the enzymes responsible for these phylogenetically diverse functions is advantageous because it provides direct functional information. However, no methods are available for the broad quantification of these genes from uncultured microbes characteristic of complex environments. In this study, consensus degenerate hybrid oligonucleotide primers were designed and validated to amplify both sequenced and unsequenced glycoside hydrolase genes of cellulose-degrading bacteria,
hydA
genes of fermentative bacteria,
dsrA
genes of sulfate-reducing bacteria, and
mcrA
genes of methanogenic archaea. Specificity was verified
in silico
and by cloning and sequencing of PCR products obtained from an environmental sample characterized by the target functions. The primer pairs were further adapted to quantitative PCR (Q-PCR), and the method was demonstrated on samples obtained from two sulfate-reducing bioreactors treating mine drainage, one lignocellulose based and the other ethanol fed. As expected, the Q-PCR analysis revealed that the lignocellulose-based bioreactor contained higher numbers of cellulose degraders, fermenters, and methanogens, while the ethanol-fed bioreactor was enriched in sulfate reducers. The suite of primers developed represents a significant advance over prior work, which, for the most part, has targeted only pure cultures or has suffered from low specificity. Furthermore, ensuring the suitability of the primers for Q-PCR provided broad quantitative access to genes that drive critical anaerobic catalytic processes.
Biofilm Formation by
Campylobacter jejuni
Is Increased under Aerobic Conditions
Authors: Reuter Mark , Mallett Arthur , Pearson Bruce M. , van Vliet Arnoud H. M. 
Published: February 2010

ABSTRACT

The microaerophilic human pathogen
Campylobacter jejuni
is the leading cause of food-borne bacterial gastroenteritis in the developed world. During transmission through the food chain and the environment, the organism must survive stressful environmental conditions, particularly high oxygen levels. Biofilm formation has been suggested to play a role in the environmental survival of this organism. In this work we show that
C. jejuni
NCTC 11168 biofilms developed more rapidly under environmental and food-chain-relevant aerobic conditions (20% O
2
) than under microaerobic conditions (5% O
2
, 10% CO
2
), although final levels of biofilms were comparable after 3 days. Staining of biofilms with Congo red gave results similar to those obtained with the commonly used crystal violet staining. The level of biofilm formation by nonmotile aflagellate strains was lower than that observed for the motile flagellated strain but nonetheless increased under aerobic conditions, suggesting the presence of flagellum-dependent and flagellum-independent mechanisms of biofilm formation in
C. jejuni
. Moreover, preformed biofilms shed high numbers of viable
C. jejuni
cells into the culture supernatant independently of the oxygen concentration, suggesting a continuous passive release of cells into the medium rather than a condition-specific active mechanism of dispersal. We conclude that under aerobic or stressful conditions,
C. jejuni
adapts to a biofilm lifestyle, allowing survival under detrimental conditions, and that such a biofilm can function as a reservoir of viable planktonic cells. The increased level of biofilm formation under aerobic conditions is likely to be an adaptation contributing to the zoonotic lifestyle of
C. jejuni
.
Effects of Swine Manure on Macrolide, Lincosamide, and Streptogramin B Antimicrobial Resistance in Soils
Authors: Zhou Zhi , Raskin Lutgarde , Zilles Julie L. 
Published: February 2010

ABSTRACT

Current agricultural practices involve inclusion of antimicrobials in animal feed and result in manure containing antimicrobials and antimicrobial-resistant microorganisms. This work evaluated the effects of land application of swine manure on the levels of tetracycline, macrolide, and lincosamide antimicrobials and on macrolide, lincosamide, and streptogramin B (MLS
B
) resistance in field soil samples and laboratory soil batch tests. MLS
B
and tetracycline antimicrobials were quantified after solid-phase extraction using liquid chromatography-tandem mass spectrometry. The prevalence of the ribosomal modification responsible for MLS
B
resistance in the same samples was quantified using fluorescence
in situ
hybridization. Macrolide antimicrobials were not detected in soil samples, while tetracyclines were detected, suggesting that the latter compounds persist in soil. No significant differences in ribosomal methylation or presumed MLS
B
resistance were observed when amended and unamended field soils were compared, although a transient (<20-day) increase was observed in most batch tests.
Clostridium
cluster XIVa accounted for the largest fraction of resistant bacteria identified in amended soils. Overall, this study did not detect a persistent increase in the prevalence of MLS
B
resistance due to land application of treated swine manure.
Enhancement of Enteric Adenovirus Cultivation by Viral Transactivator Proteins
Authors: Kim Misoon , Lim Mi Young , Ko GwangPyo 
Published: February 2010

ABSTRACT
Human enteric adenoviruses (HAdVs; serotypes 40 and 41) are important waterborne and food-borne pathogens. However, HAdVs are fastidious, are difficult to cultivate, and do not produce a clear cytopathic effect during cell culture within a reasonable time. Thus, we examined whether the viral transactivator proteins cytomegalovirus (CMV) IE1 and hepatitis B virus (HBV) X promoted the multiplication of HAdVs. Additionally, we constructed a new 293 cell line expressing CMV IE1 protein for cultivation assays. We analyzed the nucleic acid sequences of the promoter regions of both E1A and hexon genes, which are considered to be the most important regions for HAdV replication. Expression of either HBV X or CMV IE1 protein significantly increased the promoter activities of E1A and hexon genes of HAdVs by as much as 14-fold during cell cultivation. The promotion of HAdV expression was confirmed by increased levels of both adenoviral DNA and mRNA expression. Finally, the newly developed 293 cell line expressing CMV IE1 protein showed an increase in viral DNA ranging from 574% to 619% compared with the conventional 293 cell line. These results suggest that the newly constructed cell line could be useful for efficient cultivation and research of fastidious HAdVs.
Requirement of a Mitogen-Activated Protein Kinase for Appressorium Formation and Penetration of Insect Cuticle by the Entomopathogenic Fungus
Beauveria bassiana
Authors: Zhang Yongjun , Zhang Jianqing , Jiang Xiaodong , Wang Guijiang , Luo Zhibing , Fan Yanhua , Wu Zengqiang , Pei Yan 
Published: February 2010

ABSTRACT

Beauveria bassiana
is an important insect-pathogenic fungus that invades insects by direct penetration of the host cuticle. To delineate the molecular mechanisms involved in fungal infection, a mitogen-activated protein kinase (MAPK) gene, Bb
mpk1
, which encodes a YERK1 family MAPK was isolated and characterized. Targeted gene disruption of Bb
mpk1
resulted in a complete loss of virulence when applied topically to host insects but did not affect growth of the fungus when conidia were injected directly into the hemocoel. Hyphae of the mutant strain growing in the insect hemocoel were unable to penetrate the cuticle growing outwards and consequently failed to sporulate on the cadaver surface. These data suggest that BbMPK1 is essential for penetration of the insect cuticle both from the outside and from the inside-out in order to escape and disperse from the host. Inactivation of BbMPK1 also caused a significant decrease in fungal adhesion to insect cuticles and eliminated their ability to form appressoria. In order to identify downstream genes regulated by BbMPK1, a suppressive subtractive hybridization (SSH) library was generated comparing mutant and wild-type transcripts isolated during appressorium formation. Thirty-one genes screened from the SSH library were determined to be expressed in the wild-type strain but either significantly reduced or not expressed in the mutant. Ten genes showed high or medium similarity to known protein encoding genes, including proteins involved in cell surface hydrophobicity, lipid metabolism, microtubule dynamics, mitochondrial electron transport, chromatin remodeling, transcription, rRNA processing, small nucleolar RNA accumulation, oxidation of aldehydes, translation, and likely other cellular processes.
Role of Extracellular DNA during Biofilm Formation by
Listeria monocytogenes
Authors: Harmsen Morten , Lappann Martin , Knøchel Susanne , Molin Søren 
Published: February 2010

ABSTRACT

Listeria monocytogenes
is a food-borne pathogen that is capable of living in harsh environments. It is believed to do this by forming biofilms, which are surface-associated multicellular structures encased in a self-produced matrix. In this paper we show that in
L. monocytogenes
extracellular DNA (eDNA) may be the only central component of the biofilm matrix and that it is necessary for both initial attachment and early biofilm formation for 41
L. monocytogenes
strains that were tested. DNase I treatment resulted in dispersal of biofilms, not only in microtiter tray assays but also in flow cell biofilm assays. However, it was also demonstrated that in a culture without eDNA, neither
Listeria
genomic DNA nor salmon sperm DNA by itself could restore the capacity to adhere. A search for additional necessary components revealed that peptidoglycan (PG), specifically
N
-acetylglucosamine (NAG), interacted with the DNA in a manner which restored adhesion. If a short DNA fragment (less than approximately 500 bp long) was added to an eDNA-free culture prior to addition of genomic or salmon sperm DNA, adhesion was prevented, indicating that high-molecular-weight DNA is required for adhesion and that the number of attachment sites on the cell surface can be saturated.
Probabilistic Model for
Listeria monocytogenes
Growth during Distribution, Retail Storage, and Domestic Storage of Pasteurized Milk
Authors: Koutsoumanis Konstantinos , Pavlis Athanasios , Nychas George-John E. , Xanthiakos Konstantinos 
Published: February 2010

ABSTRACT

A survey on the time-temperature conditions of pasteurized milk in Greece during transportation to retail, retail storage, and domestic storage and handling was performed. The data derived from the survey were described with appropriate probability distributions and introduced into a growth model of
Listeria monocytogenes
in pasteurized milk which was appropriately modified for taking into account strain variability. Based on the above components, a probabilistic model was applied to evaluate the growth of
L. monocytogenes
during the chill chain of pasteurized milk using a Monte Carlo simulation. The model predicted that, in 44.8% of the milk cartons released in the market, the pathogen will grow until the time of consumption. For these products the estimated mean total growth of
L. monocytogenes
during transportation, retail storage, and domestic storage was 0.93 log CFU, with 95th and 99th percentiles of 2.68 and 4.01 log CFU, respectively. Although based on EU regulation 2073/2005 pasteurized milk produced in Greece belongs to the category of products that do not allow the growth of
L. monocytogenes
due to a shelf life (defined by law) of 5 days, the above results show that this shelf life limit cannot prevent
L. monocytogenes
from growing under the current chill chain conditions. The predicted percentage of milk cartons—initially contaminated with 1 cell/1-liter carton—in which the pathogen exceeds the safety criterion of 100 cells/ml at the time of consumption was 0.14%. The probabilistic model was used for an importance analysis of the chill chain factors, using rank order correlation, while selected intervention and shelf life increase scenarios were evaluated. The results showed that simple interventions, such as excluding the door shelf from the domestic storage of pasteurized milk, can effectively reduce the growth of the pathogen. The door shelf was found to be the warmest position in domestic refrigerators, and it was most frequently used by the consumers for domestic storage of pasteurized milk. Furthermore, the model predicted that a combination of this intervention with a decrease of the mean temperature of domestic refrigerators by 2°C may allow an extension of pasteurized milk shelf life from 5 to 7 days without affecting the current consumer exposure to
L. monocytogenes
.
Population Diversity of
Listeria monocytogenes
LO28: Phenotypic and Genotypic Characterization of Variants Resistant to High Hydrostatic Pressure
Authors: Van Boeijen Ineke K. H. , Chavaroche Anaïs A. E. , Valderrama Wladir B. , Moezelaar Roy , Zwietering Marcel H. , Abee Tjakko 
Published: February 2010

ABSTRACT

A comparative phenotype analysis of 24
Listeria monocytogenes
LO28 stress-resistant variants obtained after high-pressure treatment was performed to assess their robustness and growth performance under a range of food-relevant conditions. In addition, genetic analysis was conducted to characterize the promoter regions and open reading frames of the class I and III transcriptional repressors CtsR and HrcA, which control production of specific sets of stress proteins. Analysis of stress survival capacity, motility, biofilm formation, and growth under various conditions showed all variants to be more resistant to pressure and heat than the wild type; however, differences among variants were observed in acid resistance, growth rate, motility, and biofilm-forming capacity. Genetic analysis revealed no variation in the genetic make-up of
hrcA
and its upstream region, but two variants had deletions in the upstream region of
ctsR
and seven variants had mutations in the
ctsR
gene itself. The results of the characterization were cluster analyzed to obtain insight into the diversity of variants. Ten unique variants and three clusters with specific features could be identified: one cluster consisting of seven variants having a mutation in the CtsR regulator gene, one cluster containing two variants with an aerobic biofilm formation capacity similar to that of the wild type, and a cluster composed of five immotile variants. The large population diversity of
L. monocytogenes
stress-resistant variants signifies the organism's genetic flexibility, which in turn may contribute to the survival and persistence of this human pathogen in food-processing environments.
Identification of Ciliate Grazers of Autotrophic Bacteria in Ammonia-Oxidizing Activated Sludge by RNA Stable Isotope Probing
Authors: Moreno Ana Maria , Matz Carsten , Kjelleberg Staffan , Manefield Mike 
Published: February 2010

ABSTRACT

It is well understood that protozoa play a major role in controlling bacterial biomass and regulating nutrient cycling in the environment. Little is known, however, about the movement of carbon from specific reduced substrates, through functional groups of bacteria, to particular clades of protozoa. In this study we first identified the active protozoan phylotypes present in activated sludge, via the construction of an rRNA-derived eukaryote clone library. Most of the sequences identified belonged to ciliates of the subclass Peritrichia and amoebae, confirming the dominance of surface-associated protozoa in the activated sludge environment. We then demonstrated that
13
C-labeled protozoan RNA can be retrieved from activated sludge amended with
13
C-labeled protozoa or
13
C-labeled
Escherichia coli
cells by using an RNA stable isotope probing (RNA-SIP) approach. Finally, we used RNA-SIP to track carbon from bicarbonate and acetate into protozoa under ammonia-oxidizing and denitrifying conditions, respectively. RNA-SIP analysis revealed that the peritrich ciliate
Epistylis galea
dominated the acquisition of carbon from bacteria with access to CO
2
under ammonia-oxidizing conditions, while there was no evidence of specific grazing on acetate consumers under denitrifying conditions.
High-Throughput Method for Rapid Induction of Prophages from Lysogens and Its Application in the Study of Shiga Toxin-Encoding
Escherichia coli
Strains
Authors: McDonald James E. , Smith Darren L. , Fogg Paul C. M. , McCarthy Alan J. , Allison Heather E. 
Published: February 2010

ABSTRACT

A high-throughput 96-well plate-based method for the rapid induction of endogenous prophages from individual bacterial strains was developed. The detection of endogenous prophages was achieved by the filtration of the culture liquor following norfloxacin induction and subsequent PCRs targeting bacteriophage-carried gene markers. The induction method was tested on 188 putative Shiga toxin (Stx)-producing
Escherichia coli
(STEC) strains and demonstrated the ability to detect both lambdoid and
stx-
carrying bacteriophages in strains for which plaques were not observed via plaque assay. Lambdoid bacteriophages were detected in 37% of the induced phage preparations via amplification of the
Q
gene, and Stx1- and Stx2-encoding phages were detected in 2 and 14% of the strains, respectively. The method therefore provided greater sensitivity for the detection of Stx and other lambdoid bacteriophage populations carried by STEC strains than that for the established method of plaque assay using bacterial indicator strains, enabling, for the first time, large-scale bacteriophage population and diversity studies.
Negative Effects of Sample Pooling on PCR-Based Estimates of Soil Microbial Richness and Community Structure
Authors: Manter Daniel K. , Weir Tiffany L. , Vivanco Jorge M. 
Published: February 2010

ABSTRACT

In this study, we examined the effect of various pooling strategies on the characterization of soil microbial community composition and phylotype richness estimates. Automated ribosomal intergenic spacer analysis (ARISA) profiles were determined from soil samples that were (i) unpooled (extracted and amplified individually), (ii) pooled prior to PCR amplification, or (iii) pooled prior to DNA extraction. Regression analyses suggest that the less even the soil microbial community (i.e., low Shannon equitability,

E
H

), the greater was the impact of either pooling strategy on microbial detection (
R
2
= 0.766). For example, at a tropical rainforest site, which had the most uneven fungal (

E
H

of 0.597) and bacterial communities (

E
H

of 0.822), the unpooled procedure detected an additional 67 fungal and 115 bacterial phylotypes relative to either of the pooled procedures. Phylotype rarity, resulting in missed detection upon pooling, differed between the fungal and bacterial communities. Fungi were typified by locally abundant but spatially rare phylotypes, and the bacteria were typified by locally rare but spatially ubiquitous phylotypes. As a result, pooling differentially influenced plot comparisons, leading to an increase in similarity for the bacterial community and a decrease in the fungal community. In conclusion, although pooling reduces sample numbers and variability, it could mask a significant portion of the detectable microbial community, particularly for fungi due to their higher spatial heterogeneity.
Activated Alleles of the Schizosaccharomyces pombegpa2+Gα Gene Identify Residues Involved in GDP-GTP Exchange
Authors: Ivey F. Douglas , Taglia Francis X. , Yang Fan , Lander Matthew M. , Kelly David A. , Hoffman Charles S. 
Published: February 2010

ABSTRACTTheSchizosaccharomyces pombeglucose/cyclic AMP (cAMP) signaling pathway includes the Gpa2-Git5-Git11 heterotrimeric G protein, whose Gpa2 Gα subunit directly binds to and activates adenylate cyclase in response to signaling from the Git3 G protein-coupled receptor. To study intrinsic and extrinsic regulation of Gpa2, we developed a plasmid-based screen to identify mutationally activatedgpa2alleles that bypass the loss of the Git5-Git11 Gβγ dimer to repress transcription of the glucose-regulatedfbp1+gene. Fifteen independently isolated mutations alter 11 different Gpa2 residues, with all but one conferring a receptor-independent activated phenotype upon integration into thegpa2+chromosomal locus. Biochemical characterization of three activated Gpa2 proteins demonstrated an increased GDP-GTP exchange rate that would explain the mechanism of activation. Interestingly, the amino acid altered in the Gpa2(V90A) exchange rate mutant protein is in a region of Gpa2 with no obvious role in Gα function, thus extending our understanding of Gα protein structure-function relationships.
F-Actin Dynamics in Neurospora crassa
Authors: Berepiki Adokiye , Lichius Alexander , Shoji Jun-Ya , Tilsner Jens , Read Nick D. 
Published: February 2010

ABSTRACT

This study demonstrates the utility of Lifeact for the investigation of actin dynamics in
Neurospora crassa
and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential
Saccharomyces cerevisiae
actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in
N. crassa
without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth.
Ste12 and Ste12-Like Proteins, Fungal Transcription Factors Regulating Development and Pathogenicity
Authors: Wong Sak Hoi Joanne , Dumas Bernard 
Published: February 2010

ABSTRACT

Ste12 and Ste12-like proteins are transcription factors found exclusively in the fungal kingdom. In the yeast model
Saccharomyces cerevisiae
, where the first member was identified, Ste12p was shown to regulate mating and invasive/pseudohyphal growth. In recent literature, there have been several reports of Ste12-like factors in multiple fungal systems, yeasts or filamentous fungi, with saprophytic or parasitic life-styles. In all these models, Ste12 and Ste12-like factors are involved in the regulation of fungal development and pathogenicity. In this review, we discuss the features, the regulation, and the role of Ste12 and Ste12-like factors by highlighting the similarities and dissimilarities that occur within this group.