bluuebunny/crossref_metadata_2025_split huggingface dataset:
Elucidation of the Molecular Basis for Arabinoxylan-Debranching Activity of a Thermostable Family GH62 α-l-Arabinofuranosidase from Streptomyces thermoviolaceus
Authors: Wang Weijun , Mai-Gisondi Galina , Stogios Peter J. , Kaur Amrit , Xu Xiaohui , Cui Hong , Turunen Ossi , Savchenko Alexei , Master Emma R. 
Published: June 2014

ABSTRACTXylan-debranching enzymes facilitate the complete hydrolysis of xylan and can be used to alter xylan chemistry. Here, the family GH62 α-l-arabinofuranosidase fromStreptomyces thermoviolaceus(SthAbf62A) was shown to have a half-life of 60 min at 60°C and the ability to cleave α-1,3l-arabinofuranose (l-Araf) from singly substituted xylopyranosyl (Xylp) backbone residues in wheat arabinoxylan; low levels of activity on arabinan as well as 4-nitrophenyl α-l-arabinofuranoside were also detected. After selective removal of α-1,3l-Arafsubstituents from disubstituted Xylpresidues present in wheat arabinoxylan, SthAbf62A could also cleave the remaining α-1,2l-Arafsubstituents, confirming the ability of SthAbf62A to remove α-l-Arafresidues that are (1→2) and (1→3) linked to monosubstituted β-d-Xylpsugars. Three-dimensional structures of SthAbf62A and its complex with xylotetraose andl-arabinose confirmed a five-bladed β-propeller fold and revealed a molecular Velcro in blade V between the β1 and β21 strands, a disulfide bond between Cys27 and Cys297, and a calcium ion coordinated in the central channel of the fold. The enzyme-arabinose complex structure further revealed a narrow and seemingly rigidl-arabinose binding pocket situated at the center of one side of the β propeller, which stabilized the arabinofuranosyl substituent through several hydrogen-bonding and hydrophobic interactions. The predicted catalytic amino acids were oriented toward this binding pocket, and the catalytic essentiality of Asp53 and Glu213 was confirmed by site-specific mutagenesis. Complex structures with xylotetraose revealed a shallow cleft for xylan backbone binding that is open at both ends and comprises multiple binding subsites above and flanking thel-arabinose binding pocket.
Effect of Microstructure on Population Growth Parameters of Escherichia coli in Gelatin-Dextran Systems
Authors: Boons Kathleen , Noriega Estefanía , Van den Broeck Rob , David Charlotte C. , Hofkens Johan , Van Impe Jan F. 
Published: June 2014

ABSTRACT

Current literature acknowledges the effect of food structure on bacterial dynamics. Most studies introduce this “structure” factor using a single gelling agent, resulting in a homogeneous environment, whereas in practice most food products are heterogeneous. Therefore, this study focuses on heterogeneous protein-polysaccharide mixtures, based on gelatin and dextran. These mixtures show phase separation, leading to a range of heterogeneous microstructures by adjusting relative concentrations of both gelling agents. Based on confocal microscope observations, the growth of
Escherichia coli
in gelatin-dextran systems was observed to occur in the dextran phase. To find a relation between microscopic and population behavior, growth experiments were performed in binary and singular gelatin-dextran systems and culture broth at 23.5°C, with or without adding 2.9% (wt/vol) NaCl. The Baranyi and Roberts growth model was fitted to the experimental data and parameter estimates were statistically compared. For salted binary mixtures, a decrease in the population maximum cell density was observed with increasing gelatin concentration. In this series, for one type of microstructure, i.e., a gelatin matrix phase with a disperse dextran phase, the maximum cell density decreased with decreasing percentage of dextran phase. However, this relation no longer held when other types of microstructure were observed. Compared to singular systems, adding a second gelling agent in the presence of NaCl had an effect on population lag phases and maximum cell densities. For unsalted media, the growth parameters of singular and binary mixtures were comparable. Introducing this information into mathematical models leads to more reliable growth predictions and enhanced food safety.
Analysis of Strains Lacking Known Osmolyte Accumulation Mechanisms Reveals Contributions of Osmolytes and Transporters to Protection against Abiotic Stress
Authors: Murdock Lindsay , Burke Tangi , Coumoundouros Chelsea , Culham Doreen E. , Deutch Charles E. , Ellinger James , Kerr Craig H. , Plater Samantha M. , To Eric , Wright Geordie , Wood Janet M. 
Published: June 2014

ABSTRACT

Osmolyte accumulation and release can protect cells from abiotic stresses. In
Escherichia coli
, known mechanisms mediate osmotic stress-induced accumulation of K
+
glutamate, trehalose, or zwitterions like glycine betaine. Previous observations suggested that additional osmolyte accumulation mechanisms (OAMs) exist and their impacts may be abiotic stress specific. Derivatives of the uropathogenic strain CFT073 and the laboratory strain MG1655 lacking known OAMs were created. CFT073 grew without osmoprotectants in minimal medium with up to 0.9 M NaCl. CFT073 and its OAM-deficient derivative grew equally well in high- and low-osmolality urine pools. Urine-grown bacteria did not accumulate large amounts of known or novel osmolytes. Thus, CFT073 showed unusual osmotolerance and did not require osmolyte accumulation to grow in urine. Yeast extract and brain heart infusion stimulated growth of the OAM-deficient MG1655 derivative at high salinity. Neither known nor putative osmoprotectants did so. Glutamate and glutamine accumulated after growth with either organic mixture, and no novel osmolytes were detected. MG1655 derivatives retaining individual OAMs were created. Their abilities to mediate osmoprotection were compared at 15°C, 37°C without or with urea, and 42°C. Stress protection was not OAM specific, and variations in osmoprotectant effectiveness were similar under all conditions. Glycine betaine and dimethylsulfoniopropionate (DMSP) were the most effective. Trimethylamine-
N
-oxide (TMAO) was a weak osmoprotectant and a particularly effective urea protectant. The effectiveness of glycine betaine, TMAO, and proline as osmoprotectants correlated with their preferential exclusion from protein surfaces, not with their propensity to prevent protein denaturation. Thus, their effectiveness as stress protectants correlated with their ability to rehydrate the cytoplasm.
Norovirus Transmission between Hands, Gloves, Utensils, and Fresh Produce during Simulated Food Handling
Authors: Rönnqvist M. , Aho E. , Mikkelä A. , Ranta J. , Tuominen P. , Rättö M. , Maunula L. 
Published: June 2014

ABSTRACT

Human noroviruses (HuNoVs), a leading cause of food-borne gastroenteritis worldwide, are easily transferred via ready-to-eat (RTE) foods, often prepared by infected food handlers. In this study, the transmission of HuNoV and murine norovirus (MuNoV) from virus-contaminated hands to latex gloves during gloving, as well as from virus-contaminated donor surfaces to recipient surfaces after simulated preparation of cucumber sandwiches, was inspected. Virus transfer was investigated by swabbing with polyester swabs, followed by nucleic acid extraction from the swabs with a commercial kit and quantitative reverse transcription-PCR. During gloving, transfer of MuNoV dried on the hand was observed 10/12 times. HuNoV, dried on latex gloves, was disseminated to clean pairs of gloves 10/12 times, whereas HuNoV without drying was disseminated 11/12 times. In the sandwich-preparing simulation, both viruses were transferred repeatedly to the first recipient surface (left hand, cucumber, and knife) during the preparation. Both MuNoV and HuNoV were transferred more efficiently from latex gloves to cucumbers (1.2% ± 0.6% and 1.5% ± 1.9%) than vice versa (0.7% ± 0.5% and 0.5% ± 0.4%). We estimated that transfer of at least one infective HuNoV from contaminated hands to the sandwich prepared was likely to occur if the hands of the food handler contained 3 log
10
or more HuNoVs before gloving. Virus-contaminated gloves were estimated to transfer HuNoV to the food servings more efficiently than a single contaminated cucumber during handling. Our results indicate that virus-free food ingredients and good hand hygiene are needed to prevent HuNoV contamination of RTE foods.
Wastewater Irrigation Increases the Abundance of Potentially Harmful Gammaproteobacteria in Soils in Mezquital Valley, Mexico
Authors: Broszat Melanie , Nacke Heiko , Blasi Ronja , Siebe Christina , Huebner Johannes , Daniel Rolf , Grohmann Elisabeth 
Published: June 2014

ABSTRACT

Wastewater contains large amounts of pharmaceuticals, pathogens, and antimicrobial resistance determinants. Only a little is known about the dissemination of resistance determinants and changes in soil microbial communities affected by wastewater irrigation. Community DNAs from Mezquital Valley soils under irrigation with untreated wastewater for 0 to 100 years were analyzed by quantitative real-time PCR for the presence of
sul
genes, encoding resistance to sulfonamides. Amplicon sequencing of bacterial 16S rRNA genes from community DNAs from soils irrigated for 0, 8, 10, 85, and 100 years was performed and revealed a 14% increase of the relative abundance of
Proteobacteria
in rainy season soils and a 26.7% increase in dry season soils for soils irrigated for 100 years with wastewater. In particular,
Gammaproteobacteria
, including potential pathogens, such as
Pseudomonas
,
Stenotrophomonas
, and
Acinetobacter
spp., were found in wastewater-irrigated fields. 16S rRNA gene sequencing of 96 isolates from soils irrigated with wastewater for 100 years (48 from dry and 48 from rainy season soils) revealed that 46% were affiliated with the
Gammaproteobacteria
(mainly potentially pathogenic
Stenotrophomonas
strains) and 50% with the
Bacilli
, whereas all 96 isolates from rain-fed soils (48 from dry and 48 from rainy season soils) were affiliated with the
Bacilli
. Up to six types of antibiotic resistance were found in isolates from wastewater-irrigated soils; sulfamethoxazole resistance was the most abundant (33.3% of the isolates), followed by oxacillin resistance (21.9% of the isolates). In summary, we detected an increase of potentially harmful bacteria and a larger incidence of resistance determinants in wastewater-irrigated soils, which might result in health risks for farm workers and consumers of wastewater-irrigated crops.
LacR Is a Repressor of
lacABCD
and LacT Is an Activator of
lacTFEG
, Constituting the
lac
Gene Cluster in Streptococcus pneumoniae
Authors: Afzal Muhammad , Shafeeq Sulman , Kuipers Oscar P. 
Published: June 2014

ABSTRACT

Comparison of the transcriptome of
Streptococcus pneumoniae
strain D39 grown in the presence of either lactose or galactose with that of the strain grown in the presence of glucose revealed the elevated expression of various genes and operons, including the
lac
gene cluster, which is organized into two operons, i.e.,
lac
operon I (
lacABCD
) and
lac
operon II (
lacTFEG
). Deletion of the DeoR family transcriptional regulator
lacR
that is present downstream of the
lac
gene cluster revealed elevated expression of
lac
operon I even in the absence of lactose. This suggests a function of LacR as a transcriptional repressor of
lac
operon I, which encodes enzymes involved in the phosphorylated tagatose pathway in the absence of lactose or galactose. Deletion of
lacR
did not affect the expression of
lac
operon II, which encodes a lactose-specific phosphotransferase. This finding was further confirmed by β-galactosidase assays with P
lacA-lacZ
and P
lacT-lacZ
in the presence of either lactose or glucose as the sole carbon source in the medium. This suggests the involvement of another transcriptional regulator in the regulation of
lac
operon II, which is the BglG-family transcriptional antiterminator LacT. We demonstrate the role of LacT as a transcriptional activator of
lac
operon II in the presence of lactose and CcpA-independent regulation of the
lac
gene cluster in
S. pneumoniae
.
Genetic Characterization of Plasmid-Associated Triphenylmethane Reductase in Listeria monocytogenes
Authors: Dutta Vikrant , Elhanafi Driss , Osborne Jason , Martinez Mira Rakic , Kathariou Sophia 
Published: June 2014

ABSTRACT

The enzyme triphenylmethane reductase (TMR) reduces toxic triphenylmethane dyes into colorless, nontoxic derivatives, and TMR-producing microorganisms have been proposed as bioremediation tools. Analysis of the genome of
Listeria monocytogenes
H7858 (1998-1999 hot dog outbreak) revealed that the plasmid (pLM80) of this strain harboring a gene cassette (
bcrABC
) conferring resistance to benzalkonium chloride (BC) and other quaternary ammonium disinfectants also harbored a gene (
tmr
) highly homologous to TMR-encoding genes from diverse Gram-negative bacteria. The pLM80-associated
tmr
was located two genes downstream of
bcrABC
as part of a putative IS
1216
composite transposon. To confirm the role of
tmr
in triphenylmethane dye detoxification, we introduced various
tmr
-harboring fragments of pLM80 in a pLM80-cured derivative of strain H7550, from the same outbreak as H7858, and assessed the resistance of the constructs to the triphenylmethane dyes crystal violet (CV) and malachite green. Transcriptional and subcloning data suggest that the regulation of TMR is complex. Constructs harboring fragments spanning
bcrABC
and
tmr
were CV resistant, and in such constructs
tmr
transcription was induced by sublethal levels of either BC or CV. However, constructs harboring only
tmr
and its upstream intergenic region could also confer resistance to CV, albeit at lower levels. Screening a panel of BC-resistant
L. monocytogenes
strains revealed that all those harboring
bcrABC
and adjacent pLM80 sequences, including
tmr
, were resistant to CV and decolorized this dye. The findings suggest a potential role of TMR as a previously unknown adaptive attribute for environmental persistence of
L. monocytogenes
.
TgaA, a VirB1-Like Component Belonging to a Putative Type IV Secretion System of Bifidobacterium bifidum MIMBb75
Authors: Guglielmetti Simone , Balzaretti Silvia , Taverniti Valentina , Miriani Matteo , Milani Christian , Scarafoni Alessio , Corona Silvia , Ciranna Alessandro , Arioli Stefania , Santala Ville , Iametti Stefania , Bonomi Francesco , Ventura Marco , Mora Diego , Karp Matti 
Published: June 2014

ABSTRACT

Bifidobacterium bifidum
MIMBb75 is a human intestinal isolate demonstrated to be interactive with the host and efficacious as a probiotic. However, the molecular biology of this microorganism is yet largely unknown. For this reason, we undertook whole-genome sequencing of
B. bifidum
MIMBb75 to identify potential genetic factors that would explain the metabolic and probiotic attributes of this bacterium. Comparative genomic analysis revealed a 45-kb chromosomal region that comprises 19 putative genes coding for a potential type IV secretion system (T4SS). Thus, we undertook the initial characterization of this genetic region by studying the putative
virB1
-like gene, named
tgaA
. Gene
tgaA
encodes a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT, cd00254.3) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP, pfam05257.4). By means of several
in vitro
assays, we experimentally confirmed that protein TgaA, consistent with its computationally assigned role, has peptidoglycan lytic activity, which is principally associated to the LT domain. Furthermore, immunofluorescence and immunogold labeling showed that the protein TgaA is abundantly expressed on the cell surface of
B. bifidum
MIMBb75. According to the literature, the T4SSs, which have not been characterized before in bifidobacteria, can have important implications for bacterial cell-to-cell communication as well as cross talk with host cells, justifying the interest for further studies aimed at the investigation of this genetic region.
Inhibition of Biofilm Formation by T7 Bacteriophages Producing Quorum-Quenching Enzymes
Authors: Pei Ruoting , Lamas-Samanamud Gisella R. 
Published: June 2014

ABSTRACT

Bacterial growth in biofilms is the major cause of recalcitrant biofouling in industrial processes and of persistent infections in clinical settings. The use of bacteriophage treatment to lyse bacteria in biofilms has attracted growing interest. In particular, many natural or engineered phages produce depolymerases to degrade polysaccharides in the biofilm matrix and allow access to host bacteria. However, the phage-produced depolymerases are highly specific for only the host-derived polysaccharides and may have limited effects on natural multispecies biofilms. In this study, an engineered T7 bacteriophage was constructed to encode a lactonase enzyme with broad-range activity for quenching of quorum sensing, a form of bacterial cell-cell communication via small chemical molecules (acyl homoserine lactones [AHLs]) that is necessary for biofilm formation. Our results demonstrated that the engineered T7 phage expressed the AiiA lactonase to effectively degrade AHLs from many bacteria. Addition of the engineered T7 phage to mixed-species biofilms containing
Pseudomonas aeruginosa
and
Escherichia coli
resulted in inhibition of biofilm formation. Such quorum-quenching phages that can lyse host bacteria and express quorum-quenching enzymes to affect diverse bacteria in biofilm communities may become novel antifouling and antibiofilm agents in industrial and clinical settings.
Iron Triggers λSo Prophage Induction and Release of Extracellular DNA in Shewanella oneidensis MR-1 Biofilms
Authors: Binnenkade Lucas , Teichmann Laura , Thormann Kai M. 
Published: June 2014

ABSTRACT

Prophages are ubiquitous elements within bacterial chromosomes and affect host physiology and ecology in multiple ways. We have previously demonstrated that phage-induced lysis is required for extracellular DNA (eDNA) release and normal biofilm formation in
Shewanella oneidensis
MR-1. Here, we investigated the regulatory mechanisms of prophage λSo spatiotemporal induction in biofilms. To this end, we used a functional fluorescence fusion to monitor λSo activation in various mutant backgrounds and in response to different physiological conditions. λSo induction occurred mainly in a subpopulation of filamentous cells in a strictly RecA-dependent manner, implicating oxidative stress-induced DNA damage as the major trigger. Accordingly, mutants affected in the oxidative stress response (Δ
oxyR
) or iron homeostasis (Δ
fur
) displayed drastically increased levels of phage induction and abnormal biofilm formation, while planktonic cells were not or only marginally affected. To further investigate the role of oxidative stress, we performed a mutant screen and identified two independent amino acid substitutions in OxyR (T104N and L197P) that suppress induction of λSo by hydrogen peroxide (H
2
O
2
). However, λSo induction was not suppressed in biofilms formed by both mutants, suggesting a minor role of intracellular H
2
O
2
in this process. In contrast, addition of iron to biofilms strongly enhanced λSo induction and eDNA release, while both processes were significantly suppressed at low iron levels, strongly indicating that iron is the limiting factor. We conclude that uptake of iron during biofilm formation triggers λSo-mediated lysis of a subpopulation of cells, likely by an increase in iron-mediated DNA damage sensed by RecA.
The Functional Structure of Central Carbon Metabolism in Pseudomonas putida KT2440
Authors: Sudarsan Suresh , Dethlefsen Sarah , Blank Lars M. , Siemann-Herzberg Martin , Schmid Andreas 
Published: June 2014

ABSTRACT

What defines central carbon metabolism? The classic textbook scheme of central metabolism includes the Embden-Meyerhof-Parnas (EMP) pathway of glycolysis, the pentose phosphate pathway, and the citric acid cycle. The prevalence of this definition of central metabolism is, however, equivocal without experimental validation. We address this issue using a general experimental approach that combines the monitoring of transcriptional and metabolic flux changes between steady states on alternative carbon sources. This approach is investigated by using the model bacterium
Pseudomonas putida
with glucose, fructose, and benzoate as carbon sources. The catabolic reactions involved in the initial uptake and metabolism of these substrates are expected to show a correlated change in gene expressions and metabolic fluxes. However, there was no correlation for the reactions linking the 12 biomass precursor molecules, indicating a regulation mechanism other than mRNA synthesis for central metabolism. This result substantiates evidence for a (re)definition of central carbon metabolism including all reactions that are bound to tight regulation and transcriptional invariance. Contrary to expectations, the canonical Entner-Doudoroff and EMP pathways
sensu stricto
are not a part of central carbon metabolism in
P. putida
, as they are not regulated differently from the aromatic degradation pathway. The regulatory analyses presented here provide leads on a qualitative basis to address the use of alternative carbon sources by deregulation and overexpression at the transcriptional level, while rate improvements in central carbon metabolism require careful adjustment of metabolite concentrations, as regulation resides to a large extent in posttranslational and/or metabolic regulation.
Interplay between Candida albicans and the Antimicrobial Peptide Armory
Authors: Swidergall Marc , Ernst Joachim F. 
Published: June 2014

ABSTRACT

Antimicrobial peptides (AMPs) are key elements of innate immunity, which can directly kill multiple bacterial, viral, and fungal pathogens. The medically important fungus
Candida albicans
colonizes different host niches as part of the normal human microbiota. Proliferation of
C. albicans
is regulated through a complex balance of host immune defense mechanisms and fungal responses. Expression of AMPs against pathogenic fungi is differentially regulated and initiated by interactions of a variety of fungal pathogen-associated molecular patterns (PAMPs) with pattern recognition receptors (PRRs) on human cells. Inflammatory signaling and other environmental stimuli are also essential to control fungal proliferation and to prevent parasitism. To persist in the host,
C. albicans
has developed a three-phase AMP evasion strategy, including secretion of peptide effectors, AMP efflux pumps, and regulation of signaling pathways. These mechanisms prevent
C. albicans
from the antifungal activity of the major AMP classes, including cathelicidins, histatins, and defensins leading to a basal resistance. This minireview summarizes human AMP attack and
C. albicans
resistance mechanisms and current developments in the use of AMPs as antifungal agents.