bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development and Application of a Method for Counterselectable In-Frame Deletion in
Clostridium perfringens
Authors: Nariya Hirofumi , Miyata Shigeru , Suzuki Motoo , Tamai Eiji , Okabe Akinobu 
Published: December 2010

ABSTRACT

Many pathogenic clostridial species produce toxins and enzymes. To facilitate genome-wide identification of virulence factors and biotechnological application of their useful products, we have developed a markerless in-frame deletion method for
Clostridium perfringens
which allows efficient counterselection and multiple-gene disruption. The system comprises a
galKT
gene disruptant and a suicide
galK
plasmid into which two fragments of a target gene for in-frame deletion are cloned. The system was shown to be accurate and simple by using it to disrupt the alpha-toxin gene of the organism. It was also used to construct of two different virulence-attenuated strains, ΗΝ1303 and HN1314: the former is a disruptant of the
virRS
operon, which regulates the expression of virulence factors, and the latter is a disruptant of the six genes encoding the α, θ, and κ toxins; a clostripain-like protease; a 190-kDa secretory protein; and a putative cell wall lytic endopeptidase. Comparison of the two disruptants in terms of growth ability and the background levels of secreted proteins showed that HN1314 is more useful than ΗΝ1303 as a host for the large-scale production of recombinant proteins.
Characterization of a Phosphotriesterase-Like Lactonase from
Sulfolobus solfataricus
and Its Immobilization for Disruption of Quorum Sensing
Authors: Ng Filomena S. W. , Wright Daniel M. , Seah Stephen Y. K. 
Published: December 2010

ABSTRACT

SsoPox, a bifunctional enzyme with organophosphate hydrolase and
N
-acyl homoserine lactonase activities from the hyperthermophilic archaeon
Sulfolobus solfataricus
, was overexpressed and purified from recombinant
Pseudomonas putida
KT2440 with a yield of 9.4 mg of protein per liter of culture. The enzyme has a preference for
N
-acyl homoserine lactones (AHLs) with acyl chain lengths of at least 8 carbon atoms, mainly due to lower

K
m

values for these substrates. The highest specificity constant obtained was for
N
-3-oxo-decanoyl homoserine lactone (
k
cat
/

K
m

= 5.5 × 10
3
M
−1
·s
−1
), but SsoPox can also degrade
N
-butyryl homoserine lactone (C
4
-HSL) and
N
-oxo-dodecanoyl homoserine lactone (oxo-C
12
-HSL), which are important for quorum sensing in our
Pseudomonas aeruginosa
model system. When
P. aeruginosa
PAO1 cultures were grown in the presence of SsoPox-immobilized membranes, the production of C
4
-HSL- and oxo-C
12
-HSL-regulated virulence factors, elastase, protease, and pyocyanin were significantly reduced. This is the first demonstration that immobilized quorum-quenching enzymes can be used to attenuate the production of virulence factors controlled by quorum-sensing signals.
Cytoplasmic and Periplasmic Proteomic Signatures of Exponentially Growing Cells of the Psychrophilic Bacterium
Pseudoalteromonas haloplanktis
TAC125
Authors: Wilmes Boris , Kock Holger , Glagla Susanne , Albrecht Dirk , Voigt Birgit , Markert Stephanie , Gardebrecht Antje , Bode Rüdiger , Danchin Antoine , Feller Georges , Hecker Michael , Schweder Thomas 
Published: December 2010

ABSTRACT

The psychrophilic model bacterium
Pseudoalteromonas haloplanktis
is characterized by remarkably fast growth rates under low-temperature conditions in a range from 5°C to 20°C. In this study the proteome of cellular compartments, the cytoplasm and periplasm, of
P. haloplanktis
strain TAC125 was analyzed under exponential growth conditions at a permissive temperature of 16°C. By means of two-dimensional protein gel electrophoresis and mass spectrometry, a first inventory of the most abundant cytoplasmic and periplasmic proteins expressed in a peptone-supplemented minimal medium was established. By this approach major enzymes of the amino acid catabolism of this marine bacterium could be functionally deduced. The cytoplasmic proteome showed a predominance of amino acid degradation pathways and tricarboxylic acid (TCA) cycle enzymes but also the protein synthesis machinery. Furthermore, high levels of cold acclimation and oxidative stress proteins could be detected at this moderate growth temperature. The periplasmic proteome was characterized by a significant abundance of transporters, especially of highly expressed putative TonB-dependent receptors. This high capacity for protein synthesis, efficient amino acid utilization, and substrate transport may contribute to the fast growth rates of the copiotrophic bacterium
P. haloplanktis
in its natural environments.
Development and Application of a Tool To Assess Laboratory Hygiene in Contained-Use Facilities
Authors: Rutjes S. A. , Lodder-Verschoor F. , Tijssen J. P. , de Roda Husman A. M. 
Published: December 2010

ABSTRACT
To gain information on laboratory hygiene in contained-use laboratories, a method was developed to study the presence of microorganisms on laboratory equipment. Focusing detection on genetically modified organisms (GMOs) containing the universal M13 primer binding sites enabled the detection of a broad range of GMOs using a single PCR. Swabbing surfaces in three different contained-use laboratories led to detection of M13-containing PCR products in 26 out of 34 swabs. Most sequences (up to five per sample) were detected in swabs from the centrifuge and sink, followed by swabs taken from the bin and incubator (up to four sequences per sample). The obtained sequences varied in length from 171 nucleotides (nt) to 878 nt. In most cases, sequences were only partially similar to sequences published in GenBank. The lengths of the regions with high similarity varied from 94 nt to 795 nt, and these similarities ranged from 81% to 100%. Similarities with more than one sequence were commonly found, complicating the identification of detected sequences. Nonetheless, 84% of the detected sequences were actually handled in the laboratory at the time of sampling. This demonstrates that the method may be used as a quality control tool to assess the efficacy of decontamination and cleaning of commonly used surfaces, such as laboratory benches, freezer doors, and centrifuge rotors, without prior knowledge of the identity or characteristics of the GMOs.
Metagenomic Analysis of the Viral Communities in Fermented Foods
Authors: Park Eun-Jin , Kim Kyoung-Ho , Abell Guy C. J. , Kim Min-Soo , Roh Seong Woon , Bae Jin-Woo 
Published: December 2010

ABSTRACT

Viruses are recognized as the most abundant biological components on Earth, and they regulate the structure of microbial communities in many environments. In soil and marine environments, microorganism-infecting phages are the most common type of virus. Although several types of bacteriophage have been isolated from fermented foods, little is known about the overall viral assemblages (viromes) of these environments. In this study, metagenomic analyses were performed on the uncultivated viral communities from three fermented foods, fermented shrimp, kimchi, and sauerkraut. Using a high-throughput pyrosequencing technique, a total of 81,831, 70,591 and 69,464 viral sequences were obtained from fermented shrimp, kimchi and sauerkraut, respectively. Moreover, 37 to 50% of these sequences showed no significant hit against sequences in public databases. There were some discrepancies between the prediction of bacteriophages hosts via homology comparison and bacterial distribution, as determined from 16S rRNA gene sequencing. These discrepancies likely reflect the fact that the viral genomes of fermented foods are poorly represented in public databases. Double-stranded DNA viral communities were amplified from fermented foods by using a linker-amplified shotgun library. These communities were dominated by bacteriophages belonging to the viral order
Caudovirales
(i.e.,
Myoviridae
,
Podoviridae
, and
Siphoviridae
). This study indicates that fermented foods contain less complex viral communities than many other environmental habitats, such as seawater, human feces, marine sediment, and soil.
Genome Sequence and Characterization of the
Tsukamurella
Bacteriophage TPA2
Authors: Petrovski Steve , Seviour Robert J. , Tillett Daniel 
Published: December 2010

ABSTRACT

The formation of stable foam in activated sludge plants is a global problem for which control is difficult. These foams are often stabilized by hydrophobic mycolic acid-synthesizing
Actinobacteria
, among which are
Tsukamurella
spp. This paper describes the isolation from activated sludge of the novel double-stranded DNA phage TPA2. This polyvalent
Siphoviridae
family phage is lytic for most
Tsukamurella
species. Whole-genome sequencing reveals that the TPA2 genome is circularly permuted (61,440 bp) and that 70% of its sequence is novel. We have identified 78 putative open reading frames, 95 pairs of inverted repeats, and 6 palindromes. The TPA2 genome has a modular gene structure that shares some similarity to those of
Mycobacterium
phages. A number of the genes display a mosaic architecture, suggesting that the TPA2 genome has evolved at least in part from genetic recombination events. The genome sequence reveals many novel genes that should inform any future discussion on
Tsukamurella
phage evolution.
Nationwide Groundwater Surveillance of Noroviruses in South Korea, 2008
Authors: Lee Sung-Geun , Jheong Weon-Hwa , Suh Chang-Il , Kim Sang-Hyun , Lee Joong-Bok , Jeong Yong-Seok , Ko GwangPyo , Jang Kyung Lib , Lee Gyu-Cheol , Paik Soon-Young 
Published: December 2010

ABSTRACTTo inspect the norovirus contamination of groundwater in South Korea, a nationwide study was performed in the summer (June to August) and winter (October to December) of 2008. Three-hundred sites designated by the government ministry were inspected. Water samples were collected for analysis of water quality, microorganism content, and viral content. Water quality was assessed by temperature, pH, turbidity, residual chlorine, and nitrite nitrogen content. Microorganism contents were analyzed bacteria, total coliforms,Escherichia coli, and bacteriophage. Virus analyses included panenterovirus and norovirus. Two primer sets were used for the detection of norovirus genotypes GI and GII, respectively. Of 300 samples, 65 (21.7%) were norovirus positive in the summer and in 52 (17.3%) were norovirus positive in the winter. The genogroup GI noroviruses that were identified were GI-1, GI-2, GI-3, GI-4, GI-5, GI-6, and GI-8 genotypes; those in the GII genogroup were GII-4 and GII-Yuri genotypes. The analytic data showed correlative relationships between the norovirus detection rate and the following parameters: water temperature and turbidity in physical-chemical parameters and somatic phage in microbial parameters. It is necessary to periodically monitor waterborne viruses that frequently cause epidemic food poisoning in South Korea for better public health and sanitary conditions.
Novel Antibacterial Proteins from the Microbial Communities Associated with the Sponge
Cymbastela concentrica
and the Green Alga
Ulva australis
Authors: Yung Pui Yi , Burke Catherine , Lewis Matt , Kjelleberg Staffan , Thomas Torsten 
Published: December 2010

ABSTRACT
The functional metagenomic screening of the microbial communities associated with a temperate marine sponge and a green alga identified three novel hydrolytic enzymes with antibacterial activities. The results suggest that uncultured alpha- and gammaproteobacteria contain new classes of proteins that may be a source of antibacterial agents.
Identification of Candidate Carrier Proteins for Surface Display on
Lactococcus lactis
by Theoretical and Experimental Analyses of the Surface Proteome
Authors: Berlec Aleš , Zadravec Petra , Jevnikar Zala , Štrukelj Borut 
Published: December 2010

ABSTRACT

Lactococcus lactis
is a lactic acid bacterium of proven safety for use in human oral applications. For this purpose, surface display of recombinant proteins is important, and new approaches for it are being sought. Analysis of the bacterial surface proteome is essential in identifying new candidate carrier proteins for surface display. We have made two different predictions of surface-associated proteins of
L. lactis
MG1363 by using Augur and LocateP software, which yielded 666 and 648 proteins, respectively. Surface proteins of
L. lactis
NZ9000, a derivative of MG1363, were identified by using a proteomics approach. The surface proteins were cleaved from intact bacteria, and the resulting peptides were identified by mass spectrometry. The latter approach yielded 80 proteins, 34 of which were not predicted by either software. Of the 80 proteins, 7 were selected for further study. These were cloned in frame with a C-terminal hexahistidine tag and overexpressed in
L. lactis
NZ9000 using nisin-controlled expression. Proteins of correct molecular weight carrying a hexahistidine tag were detected. Their surface localization was confirmed with flow cytometry. Basic membrane protein A (BmpA) was exposed at the highest level. To test BmpA as a candidate carrier protein, the hexahistidine tag was replaced by the B domain of staphylococcal protein A in the genetic construct. The B domain was displayed on the surface with BmpA as a carrier. The advantage of covalent BmpA binding was demonstrated. BmpA was thus shown to be a suitable candidate for a carrier protein in lactococcal surface display.
Bacterial Endosymbiont Localization inHyalesthes obsoletus, the Insect Vector of Bois Noir inVitis vinifera
Authors: Gonella Elena , Negri Ilaria , Marzorati Massimo , Mandrioli Mauro , Sacchi Luciano , Pajoro Massimo , Crotti Elena , Rizzi Aurora , Clementi Emanuela , Tedeschi Rosemarie , Bandi Claudio , Alma Alberto , Daffonchio Daniele 
Published: December 2010

ABSTRACTOne emerging disease of grapevine in Europe is Bois noir (BN), a phytoplasmosis caused by “CandidatusPhytoplasma solani” and spread in vineyards by the planthopperHyalesthes obsoletus(Hemiptera: Cixiidae). Here we present the first full characterization of the bacterial community of this important disease vector collected from BN-contaminated areas in Piedmont, Italy. Length heterogeneity PCR and denaturing gradient gel electrophoresis analysis targeting the 16S rRNA gene revealed the presence of a number of bacteria stably associated with the insect vector. In particular, symbiotic bacteria detected by PCR with high infection rates in adult individuals fell within the “CandidatusSulcia muelleri” cluster in theBacteroidetesand in the “CandidatusPurcelliella pentastirinorum” group in theGammaproteobacteria, both previously identified in different leafhoppers and planthoppers. A high infection rate (81%) was also shown for another symbiont belonging to theBetaproteobacteria, designated the HO1-V symbiont. Because of the low level of 16S rRNA gene identity (80%) with the closest relative, an uncharacterized symbiont of the tickHaemaphysalis longicornis, we propose the new name “CandidatusVidania fulgoroideae.” Other bacterial endosymbionts identified inH. obsoletuswere related to the intracellular bacteriaWolbachia pipientis,Rickettsiasp., and “CandidatusCardinium hertigii.” Fluorescentin situhybridization coupled with confocal laser scanning microscopy and transmission electron microscopy showed that these bacteria are localized in the gut, testicles, and oocytes. As “Ca.Sulcia” is usually reported in association with other symbiotic bacteria, we propose that inH. obsoletus, it may occur in a bipartite or even tripartite relationship between “Ca.Sulcia” and “Ca.Purcelliella,” “Ca.Vidania,” or both.
Inactivation of Murine Norovirus 1, Coliphage φX174, andBacillus fragilisPhage B40-8 on Surfaces and Fresh-Cut Iceberg Lettuce by Hydrogen Peroxide and UV Light
Authors: Li Dan , Baert Leen , De Jonghe Maarten , Van Coillie Els , Ryckeboer Jaak , Devlieghere Frank , Uyttendaele Mieke 
Published: December 2010

ABSTRACTIn this study, the inactivating properties of liquid hydrogen peroxide (L-H2O2), vaporized hydrogen peroxide (V-H2O2), UV light, and a combination of V-H2O2and UV light were tested on murine norovirus 1 (MNV-1) and bacteriophages (φX174 and B40-8) as models for human noroviruses. Disinfection of surfaces was examined on stainless steel discs based on European Standard EN 13697 (2001). For fresh-produce decontamination, a mixture of the viruses was inoculated onto shredded iceberg lettuce and treated after overnight incubation at 2°C. According to our results, L-H2O2(2.1%) was able to inactivate MNV-1 and φX174 on stainless steel discs by approximately 4 log10units within 10 min of exposure, whereas for B40-8, 15% of L-H2O2was needed to obtain a similar reduction in 10 min. Only a marginal reduction (≤1 log10unit after 5 min of exposure) by V-H2O2(2.52%) was achieved for the tested model viruses, although in combination with UV light, a 4-log10-unit decrease within 5 min of treatment was observed on stainless steel discs. Similar trends were observed for the decontamination of shredded iceberg lettuce, but the viral decline was reduced. These results demonstrated that both L-H2O2and a combination of V-H2O2and UV light can be used for norovirus inactivation on surfaces; V-H2O2(2.52%) in combination with UV light is promising for decontamination of fresh produce with much less consumption of water and disinfectant.
Influence of Two-Component Signal Transduction Systems of
Lactobacillus casei
BL23 on Tolerance to Stress Conditions
Authors: Alcántara Cristina , Revilla-Guarinos Ainhoa , Zúñiga Manuel 
Published: December 2010

ABSTRACT

Lactobacillus casei
BL23 carries 17 two-component signal transduction systems. Insertional mutations were introduced into each gene encoding the cognate response regulators, and their effects on growth under different conditions were assayed. Inactivation of systems TC01, TC06, and TC12 (LCABL_02080-LCABL_02090, LCABL_12050-LCABL_12060, and LCABL_19600-LCABL_19610, respectively) led to major growth defects under the conditions assayed.
Involvement of an Alkane Hydroxylase System of
Gordonia
sp. Strain SoCg in Degradation of Solid
n
-Alkanes
Authors: Lo Piccolo Luca , De Pasquale Claudio , Fodale Roberta , Puglia Anna Maria , Quatrini Paola 
Published: December 2010

ABSTRACT

Enzymes involved in oxidation of long-chain
n
-alkanes are still not well known, especially those in Gram-positive bacteria. This work describes the alkane degradation system of the
n
-alkane degrader actinobacterium
Gordonia
sp. strain SoCg, which is able to grow on
n
-alkanes from dodecane (C
12
) to hexatriacontane (C
36
) as the sole C source. SoCg harbors in its chromosome a single
alk
locus carrying six open reading frames (ORFs), which shows 78 to 79% identity with the alkane hydroxylase (AH)-encoding systems of other alkane-degrading actinobacteria. Quantitative reverse transcription-PCR showed that the genes encoding AlkB (alkane 1-monooxygenase), RubA3 (rubredoxin), RubA4 (rubredoxin), and RubB (rubredoxin reductase) were induced by both
n
-hexadecane and
n
-triacontane, which were chosen as representative long-chain liquid and solid
n
-alkane molecules, respectively. Biotransformation of
n
-hexadecane into the corresponding 1-hexadecanol was detected by solid-phase microextraction coupled with gas chromatography-mass spectrometry (SPME/GC-MS) analysis. The
Gordonia
SoCg
alkB
was heterologously expressed in
Escherichia coli
BL21 and in
Streptomyces coelicolor
M145, and both hosts acquired the ability to transform
n
-hexadecane into 1-hexadecanol, but the corresponding long-chain alcohol was never detected on
n
-triacontane. However, the recombinant
S. coelicolor
M145-AH, expressing the
Gordonia alkB
gene, was able to grow on
n
-triacontane as the sole C source. A SoCg
alkB
disruption mutant that is completely unable to grow on
n
-triacontane was obtained, demonstrating the role of an AlkB-type AH system in degradation of solid
n
-alkanes.
Longitudinal Evaluation of the Efficacy of Heat Treatment Procedures against
Legionella
spp. in Hospital Water Systems by Using a Flow Cytometric Assay
Authors: Allegra Severine , Grattard Florence , Girardot Françoise , Riffard Serge , Pozzetto Bruno , Berthelot Philippe 
Published: December 2010

ABSTRACT

Legionella
spp. are frequently isolated in hospital water systems. Heat shock (30 min at 70°C) is recommended by the World Health Organization to control its multiplication. The aim of the study was to evaluate retrospectively the efficacy of heat treatments by using a flow cytometry assay (FCA) able to identify viable but nonculturable (VBNC) cells. The study included
Legionella
strains (
L. pneumophila
[3 clusters] and
L. anisa
[1 cluster]) isolated from four hot water circuits of different hospital buildings in Saint-Etienne, France, during a 20-year prospective surveillance. The strains recovered from the different circuits were not epidemiologically related, but the strains isolated within a same circuit over time exhibited an identical genotypic profile. After an
in vitro
treatment of 30 min at 70°C, the mean percentage of viable cells and VBNC cells varied from 4.6% to 71.7%. The
in vitro
differences in heat sensitivity were in agreement with the observed efficacy of preventive and corrective heating measures used to control water contamination. These results suggest that
Legionella
strains can become heat resistant after heating treatments for a long time and that flow cytometry could be helpful to check the efficacy of heat treatments on
Legionella
spp. and to optimize the decontamination processes applied to water systems for the control of
Legionella
proliferation.
Identification of the Monooxygenase Gene Clusters Responsible for the Regioselective Oxidation of Phenol to Hydroquinone in Mycobacteria
Authors: Furuya Toshiki , Hirose Satomi , Osanai Hisashi , Semba Hisashi , Kino Kuniki 
Published: December 2010

ABSTRACT

Mycobacterium goodii
strain 12523 is an actinomycete that is able to oxidize phenol regioselectively at the
para
position to produce hydroquinone. In this study, we investigated the genes responsible for this unique regioselective oxidation. On the basis of the fact that the oxidation activity of
M. goodii
strain 12523 toward phenol is induced in the presence of acetone, we first identified acetone-induced proteins in this microorganism by two-dimensional electrophoretic analysis. The N-terminal amino acid sequence of one of these acetone-induced proteins shares 100% identity with that of the protein encoded by the open reading frame Msmeg_1971 in
Mycobacterium smegmatis
strain mc
2
155, whose genome sequence has been determined. Since Msmeg_1971, Msmeg_1972, Msmeg_1973, and Msmeg_1974 constitute a putative binuclear iron monooxygenase gene cluster, we cloned this gene cluster of
M. smegmatis
strain mc
2
155 and its homologous gene cluster found in
M. goodii
strain 12523. Sequence analysis of these binuclear iron monooxygenase gene clusters revealed the presence of four genes designated
mimABCD
, which encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively. When the
mimA
gene (Msmeg_1971) of
M. smegmatis
strain mc
2
155, which was also found to be able to oxidize phenol to hydroquinone, was deleted, this mutant lost the oxidation ability. This ability was restored by introduction of the
mimA
gene of
M. smegmatis
strain mc
2
155 or of
M. goodii
strain 12523 into this mutant. Interestingly, we found that these gene clusters also play essential roles in propane and acetone metabolism in these mycobacteria.