bluuebunny/crossref_metadata_2025_split huggingface dataset:
Prevention of Contamination by Xenotropic Murine Leukemia Virus-Related Virus: Susceptibility to Alcohol-Based Disinfectants and Environmental Stability
Authors: Palesch David , Khalid Mohammad , Stürzel Christina M. , Münch Jan 
Published: February 2014

ABSTRACT
Xenotropic murine leukemia virus-related virus (XMRV) represents a novel γ-retrovirus that is capable of infecting human cells and has been classified as a biosafety level 2 (BSL-2) organism. Hence, XMRV represents a potential risk for personnel in laboratories worldwide. Here, we measured the stability of XMRV and its susceptibility to alcohol-based disinfectants. To this end, we exposed an infectious XMRV reporter virus encoding a secretable luciferase to different temperatures, pH values, and disinfectants and infected XMRV-permissive Raji B cells to measure residual viral infectivity. We found that 1 min treatment of XMRV particles at 60°C is sufficient to reduce infectivity by 99.9%. XMRV infectivity was maximal at a neutral pH but was reduced by 86% at pH 4 and 99.9% at pH 10. The common hand and surface disinfectants ethanol and isopropanol as well as the cell fixation reagent paraformaldehyde abrogated XMRV infectivity entirely, as indicated by a reduction of infectivity exceeding 99.99%. Our findings provide evidence of specific means to inactivate XMRV. Their application will help to prevent unintended XMRV contamination of cell cultures in laboratories and minimize the risk for laboratory personnel and health care workers to become infected with this biosafety level 2 organism.
Dynamic Metabolic and Transcriptional Profiling of Rhodococcus sp. Strain YYL during the Degradation of Tetrahydrofuran
Authors: He Zhixing , Yao Yanlai , Lu Zhenmei , Ye Yangfang 
Published: February 2014

ABSTRACT

Although tetrahydrofuran-degrading
Rhodococcus
sp. strain YYL possesses tetrahydrofuran (THF) degradation genes similar to those of other tetrahydrofuran-degrading bacteria, a much higher degradation efficiency has been observed in strain YYL. In this study, nuclear magnetic resonance (NMR)-based metabolomics analyses were performed to explore the metabolic profiling response of strain YYL to exposure to THF. Exposure to THF slightly influenced the metabolome of strain YYL when yeast extract was present in the medium. The metabolic profile of strain YYL over time was also investigated using THF as the sole carbon source to identify the metabolites associated with high-efficiency THF degradation. Lactate, alanine, glutarate, glutamate, glutamine, succinate, lysine, trehalose, trimethylamine-
N
-oxide (TMAO), NAD
+
, and CTP were significantly altered over time in strain YYL grown in 20 mM THF. Real-time quantitative PCR (RT-qPCR) revealed changes in the transcriptional expression levels of 15 genes involved in THF degradation, suggesting that strain YYL could accumulate several disturbances in osmoregulation (trehalose, glutamate, glutamine, etc.), with reduced glycolysis levels, an accelerated tricarboxylic acid cycle, and enhanced protein synthesis. The findings obtained through
1
H NMR metabolomics analyses and the transcriptional expression of the corresponding genes are complementary for exploring the dynamic metabolic profile in organisms.
New Skin Test for Detection of Bovine Tuberculosis on the Basis of Antigen-Displaying Polyester Inclusions Produced by Recombinant Escherichia coli
Authors: Chen Shuxiong , Parlane Natalie A. , Lee Jason , Wedlock D. Neil , Buddle Bryce M. , Rehm Bernd H. A. 
Published: February 2014

ABSTRACT

The tuberculin skin test for diagnosing tuberculosis (TB) in cattle lacks specificity if animals are sensitized to environmental mycobacteria, as some antigens in purified protein derivative (PPD) prepared from
Mycobacterium bovis
are present in nonpathogenic mycobacteria. Three immunodominant TB antigens, ESAT6, CFP10, and Rv3615c, are present in members of the pathogenic
Mycobacterium tuberculosis
complex but absent from the majority of environmental mycobacteria. These TB antigens have the potential to enhance skin test specificity. To increase their immunogenicity, these antigens were displayed on polyester beads by translationally fusing them to a polyhydroxyalkanoate (PHA) synthase which mediated formation of antigen-displaying inclusions in recombinant
Escherichia coli
. The most common form of these inclusions is poly(3-hydroxybutyric acid) (PHB). The respective fusion proteins displayed on these PHB inclusions (beads) were identified using tryptic peptide fingerprinting analysis in combination with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The surface exposure and accessibility of antigens were assessed by enzyme-linked immunosorbent assay (ELISA). Polyester beads displaying all three TB antigens showed greater reactivity with TB antigen-specific antibody than did beads displaying only one TB antigen. This was neither due to cross-reactivity of antibodies with the other two antigens nor due to differences in protein expression levels between beads displaying single or three TB antigens. The triple-antigen-displaying polyester beads were used for skin testing of cattle and detected all cattle experimentally infected with
M. bovis
with no false-positive reactions observed in those sensitized to environmental mycobacteria. The results suggested applicability of TB antigen-displaying polyester inclusions as diagnostic reagents for distinguishing TB-infected from noninfected animals.
Signaling via Tumor Necrosis Factor Receptor 1 but Not Toll-Like Receptor 2 Contributes Significantly to Hydrosalpinx Development following Chlamydia muridarum Infection
Authors: Dong Xiaohua , Liu Yuanjun , Chang Xiaotong , Lei Lei , Zhong Guangming 
Published: February 2014

ABSTRACTChlamydial infection in the lower genital tract can lead to hydrosalpinx, which is accompanied by activation of both pattern recognition receptor TLR2- and inflammatory cytokine receptor TNFR1-mediated signaling pathways. In the current study, we compared the relative contributions of these two receptors to chlamydial induction of hydrosalpinx in mice. We found that mice with or without deficiencies in TLR2 or TNFR1 displayed similar time courses of live organism shedding from vaginal swabs, suggesting that these receptor-mediated signaling pathways are not required for controlling chlamydial lower genital infection. However, mice deficient in TNFR1 but not TLR2 developed significantly reduced hydrosalpinx. The decreased pathogenicity correlated with a significant reduction in interleukin-17 byin vitro-restimulated splenocytes of TNFR1-deficient mice. Although TLR2-deficient mice developed hydrosalpinx as severe as that of wild-type mice, peritoneal macrophages from mice deficient in TLR2 but not TNFR1 produced significantly reduced cytokines upon chlamydial stimulation, suggesting that reduced macrophage responses to chlamydial infection do not always lead to a reduction in hydrosalpinx. Thus, we have demonstrated that the signaling pathways triggered by the cytokine receptor TNFR1 play a more significant role in chlamydial induction of hydrosalpinx than those mediated by the pattern recognition receptor TLR2, which has laid a foundation for further revealing the chlamydial pathogenic mechanisms.
Effects of 3′-OH and 5′-PO
4
Base Mispairs and Damaged Base Lesions on the Fidelity of Nick Sealing by Deinococcus radiodurans RNA Ligase
Authors: Schmier Brad J. , Shuman Stewart 
Published: February 2014

ABSTRACT

Deinococcus radiodurans
RNA ligase (DraRnl) is the founding member of a family of end-joining enzymes encoded by diverse microbes and viruses. DraRnl ligates 3′-OH, 5′-PO
4
nicks in double-stranded nucleic acids in which the nick 3′-OH end is RNA. Here we gauge the effects of 3′-OH and 5′-PO
4
base mispairs and damaged base lesions on the rate of nick sealing. DraRnl is indifferent to the identity of the 3′-OH nucleobase, provided that it is correctly paired. With 3′-OH mispairs, the DraRnl sealing rate varies widely, with G-T and A-C mispairs being the best substrates and G-G, G-A, and A-A mispairs being the worst. DraRnl accepts 3′ A–8-oxoguanine (oxoG) to be correctly paired, while it discriminates against U-oxoG and G-oxoG mispairs. DraRnl displays high activity and low fidelity in sealing 3′-OH ends opposite an 8-oxoadenine lesion. It prefers 3′-OH adenosine when sealing opposite an abasic template site. With 5′-PO
4
mispairs, DraRnl seals a 5′ T-G mispair as well as it does a 5′ C-G pair; in most other respects, the ligation fidelity at 5′ mispairs is similar to that at 3′ mispairs. DraRnl accepts a 5′ A-oxoG end to be correctly paired, yet it is more tolerant of 5′ T-oxoG and 5′ G-oxoG mispairs than the equivalent configurations on the 3′ side of the nick. At 5′ nucleobase-abasic site nicks, DraRnl prefers to ligate when the nucleobase is a purine. The biochemical properties of DraRnl are compatible with its participation in the templated repair of RNA damage or in the sealing of filled DNA gaps that have a 3′ ribopatch.
Function and Localization Dynamics of Bifunctional Penicillin-Binding Proteins in Caulobacter crescentus
Authors: Strobel Wolfgang , Möll Andrea , Kiekebusch Daniela , Klein Kathrin E. , Thanbichler Martin 
Published: February 2014

ABSTRACT

The peptidoglycan cell wall of bacteria is a complex macromolecule composed of glycan strands that are cross-linked by short peptide bridges. Its biosynthesis involves a conserved group of enzymes, the bifunctional penicillin-binding proteins (bPBPs), which contain both a transglycosylase and a transpeptidase domain, thus being able to elongate the glycan strands and, at the same time, generate the peptide cross-links. The stalked model bacterium
Caulobacter crescentus
possesses five bPBP paralogs, named Pbp1A, PbpC, PbpX, PbpY, and PbpZ, whose function is still incompletely understood. In this study, we show that any of these proteins except for PbpZ is sufficient for growth and normal morphogenesis when expressed at native or elevated levels, whereas inactivation of all five paralogs is lethal. Growth analyses indicate a central role of PbpX in the resistance of
C. crescentus
against the noncanonical amino acid
d
-alanine. Moreover, we show that PbpX and PbpY localize to the cell division site. Their recruitment to the divisome is dependent on the essential cell division protein FtsN and likely involves interactions with FtsL and the putative peptidoglycan hydrolase DipM. The same interaction pattern is observed for Pbp1A and PbpC, although these proteins do not accumulate at midcell. Our findings demonstrate that the bPBPs of
C. crescentus
are, to a large extent, redundant and have retained the ability to interact with the peptidoglycan biosynthetic machineries responsible for cell elongation, cytokinesis, and stalk growth. Nevertheless, they may preferentially act in specific peptidoglycan biosynthetic complexes, thereby facilitating the independent regulation of distinct growth processes.
DnaJ-Promoted Binding of DnaK to Multiple Sites on σ
32
in the Presence of ATP
Authors: Noguchi Aki , Ikeda Ayami , Mezaki Moeka , Fukumori Yoshihiro , Kanemori Masaaki 
Published: February 2014

ABSTRACT

The
Escherichia coli
DnaK chaperone system is a canonical heat shock protein 70 (Hsp70) chaperone system comprising Hsp70, Hsp40, and a nucleotide exchange factor. Although Hsp40 is known to facilitate the effective binding of Hsp70 to substrates, the role of Hsp40 in Hsp70-substrate interactions has not yet been fully elucidated. Using the
E. coli
heat shock transcription factor σ
32
as a substrate in the DnaK chaperone system, we here provide new insight into the Hsp70-substrate interaction. When DnaK-σ
32
complexes formed under various conditions were analyzed by gel filtration, several DnaK-σ
32
complexes with different molecular masses were detected. The results indicated that multiple DnaK molecules simultaneously bind to σ
32
, even though it has been suggested that DnaK interacts with σ
32
at a molar ratio of 1:1. Two σ
32
mutants, L201D σ
32
and I54A σ
32
, which have reduced affinities for DnaK and DnaJ (Hsp40), respectively, were used to further characterize DnaK-σ
32
complex formation. Pulldown assays demonstrated that the affinity of I54A σ
32
for DnaK was similar to that of wild-type σ
32
in the absence of DnaJ, whereas L201D σ
32
exhibited an extremely low affinity for DnaK. However, in the presence of ATP and DnaJ, the yield of DnaK eluted with L201D σ
32
was much higher than that eluted with I54A σ
32
. These results indicate that there are multiple DnaK binding sites on σ
32
and that DnaJ strongly promotes DnaK binding to any site in the presence of ATP, regardless of the intrinsic affinity of DnaK for the site.
Impact of a Transposon Insertion in
phzF2
on the Specialized Metabolite Production and Interkingdom Interactions of Pseudomonas aeruginosa
Authors: Phelan Vanessa V. , Moree Wilna J. , Aguilar Julieta , Cornett Dale S. , Koumoutsi Alexandra , Noble Suzanne M. , Pogliano Kit , Guerrero Carlos A. , Dorrestein Pieter C. 
Published: February 2014

ABSTRACT

In microbiology, gene disruption and subsequent experiments often center on phenotypic changes caused by one class of specialized metabolites (quorum sensors, virulence factors, or natural products), disregarding global downstream metabolic effects. With the recent development of mass spectrometry-based methods and technologies for microbial metabolomics investigations, it is now possible to visualize global production of diverse classes of microbial specialized metabolites simultaneously. Using imaging mass spectrometry (IMS) applied to the analysis of microbiology experiments, we can observe the effects of mutations, knockouts, insertions, and complementation on the interactive metabolome. In this study, a combination of IMS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to visualize the impact on specialized metabolite production of a transposon insertion into a
Pseudomonas aeruginosa
phenazine biosynthetic gene,
phzF2
. The disruption of phenazine biosynthesis led to broad changes in specialized metabolite production, including loss of pyoverdine production. This shift in specialized metabolite production significantly alters the metabolic outcome of an interaction with
Aspergillus fumigatus
by influencing triacetylfusarinine production.
Characterization of the Vibrio cholerae VolA Surface-Exposed Lipoprotein Lysophospholipase
Authors: Pride Aaron C. , Guan Ziqiang , Trent M. Stephen 
Published: February 2014

ABSTRACT

Bacterial lipases play important roles in bacterial metabolism and environmental response. Our laboratory recently discovered that a novel lipoprotein lysophospholipase, VolA, localizes on the surface of the Gram-negative aquatic pathogen
Vibrio cholerae
. VolA functions to cleave exogenous lysophosphatidylcholine, freeing the fatty acid moiety for use by
V. cholerae
. This fatty acid is transported into the cell and can be used as a nutrient and, more importantly, as a way to alter the membrane architecture via incorporation into the phospholipid biosynthesis pathway. There are few examples of Gram-negative, surface-exposed lipoproteins, and VolA is unique, as it has a previously undercharacterized function in
V. cholerae
membrane remodeling. Herein, we report the biochemical characterization of VolA. We show that VolA is a canonical lipoprotein via mass spectrometry analysis and demonstrate the
in vitro
activity of VolA under a variety of conditions. Additionally, we show that VolA contains a conserved Gly-Xaa-Ser-Xaa-Gly motif typical of lipases. Interestingly, we report the observation of VolA homologs in other aquatic pathogens. An
Aeromonas hydrophila
VolA homolog complements a
V. cholerae
VolA mutant in growth on lysophosphatidylcholine as the sole carbon source and in enzymatic assays. These results support the idea that the lipase activity of surface-exposed VolA likely contributes to the success of
V. cholerae
, improving the overall adaptation and survival of the organism in different environments.
GtfA and GtfB Are Both Required for Protein
O
-Glycosylation in Lactobacillus plantarum
Authors: Lee I-Chiao , van Swam Iris I. , Tomita Satoru , Morsomme Pierre , Rolain Thomas , Hols Pascal , Kleerebezem Michiel , Bron Peter A. 
Published: February 2014

ABSTRACT

Acm2, the major autolysin of
Lactobacillus plantarum
WCFS1, was recently found to be
O
-glycosylated with
N
-acetylhexosamine, likely
N
-acetylglucosamine (GlcNAc). In this study, we set out to identify the glycosylation machinery by employing a comparative genomics approach to identify Gtf1 homologues, which are involved in fimbria-associated protein 1 (Fap1) glycosylation in
Streptococcus parasanguinis
. This
in silico
approach resulted in the identification of 6 candidate
L. plantarum
WCFS1 genes with significant homology to Gtf1, namely,
tagE1
to
tagE6
. These candidate genes were targeted by systematic gene deletion, followed by assessment of the consequences on glycosylation of Acm2. We observed a changed mobility of Acm2 on SDS-PAGE in the
tagE5E6
deletion strain, while deletion of other
tagE
genes resulted in Acm2 mobility comparable to that of the wild type. Subsequent mass spectrometry analysis of excised and in-gel-digested Acm2 confirmed the loss of glycosylation on Acm2 in the
tagE5E6
deletion mutant, whereas a lectin blot using GlcNAc-specific succinylated wheat germ agglutinin (sWGA) revealed that besides Acm2,
tagE5E6
deletion also abolished all but one other sWGA-reactive, protease-sensitive signal. Only complementation of both
tagE5
and
tagE6
restored those sWGA lectin signals, establishing that TagE5 and TagE6 are both required for the glycosylation of Acm2 as well as the vast majority of other sWGA-reactive proteins. Finally, sWGA lectin blotting experiments using a panel of 8 other
L. plantarum
strains revealed that protein glycosylation is a common feature in
L. plantarum
strains. With the establishment of these enzymes as protein glycosyltransferases, we propose to rename TagE5 and TagE6 as GtfA and GtfB, respectively.
Genome-Wide Analysis of Small RNAs Expressed by Yersinia pestis Identifies a Regulator of the Yop-Ysc Type III Secretion System
Authors: Schiano Chelsea A. , Koo Jovanka T. , Schipma Matthew J. , Caulfield Adam J. , Jafari Nadereh , Lathem Wyndham W. 
Published: February 2014

ABSTRACT

Small noncoding RNA (sRNA) molecules are integral components of the regulatory machinery for many bacterial species and are known to posttranscriptionally regulate metabolic and stress-response pathways, quorum sensing, virulence factors, and more. The Yop-Ysc type III secretion system (T3SS) is a critical virulence component for the pathogenic
Yersinia
species, and the regulation of this system is tightly controlled at each step from transcription to translocation of effectors into host cells. The contribution of sRNAs to the regulation of the T3SS in
Yersinia
has been largely unstudied, however. Previously, our lab identified a role for the sRNA chaperone protein Hfq in the regulation of components of the T3SS in the gastrointestinal pathogen
Yersinia pseudotuberculosis
. Here we present data demonstrating a similar requirement for Hfq in the closely related species
Yersinia pestis
. Through deep sequencing analysis of the
Y. pestis
sRNA-ome, we found 63 previously unidentified putative sRNAs in this species. We identified a
Yersinia
-specific sRNA, Ysr141, carried by the T3SS plasmid pCD1 that is required for the production of multiple T3SS proteins. In addition, we show that Ysr141 targets an untranslated region upstream of
yopJ
to posttranscriptionally activate the synthesis of the YopJ protein. Furthermore, Ysr141 may be an unstable and/or processed sRNA, which could contribute to its function in the regulation of the T3SS. The discovery of an sRNA that influences the synthesis of the T3SS adds an additional layer of regulation to this tightly controlled virulence determinant of
Y. pestis
.
An Innate Immunity-Regulating Virulence Determinant Is Uniquely Encoded within the Andes Virus Nucleocapsid Protein
Authors: Cimica Velasco , Dalrymple Nadine A. , Roth Eric , Nasonov Aleksandr , Mackow Erich R. 
Published: February 2014

ABSTRACT
Andes virus (ANDV) is the only hantavirus known to spread from person to person and shown to cause highly lethal hantavirus pulmonary syndrome (HPS) in patients and Syrian hamsters. Hantaviruses replicate in human endothelial cells and accomplish this by restricting the early induction of beta interferon (IFN-β)- and IFN-stimulated genes (ISGs). Our studies reveal that the ANDV nucleocapsid (N) protein uniquely inhibits IFN signaling responses directed by cytoplasmic double-stranded RNA (dsRNA) sensors RIG-I and MDA5. In contrast, N proteins from Sin Nombre, New York-1, and Prospect Hill hantaviruses had no effect on RIG-I/MDA5-directed transcriptional responses from IFN-β-, IFN-stimulated response element (ISRE)-, or κB-containing promoters. Ablating a potential S-segment nonstructural open reading frame (ORF) (NSs) within the ANDV plasmid expressing N protein failed to alter IFN regulation by ANDV N protein. Further analysis demonstrated that expressing the ANDV N protein inhibited downstream IFN pathway activation directed by MAVS, TBK1, and IκB kinase ε (IKKε) but failed to inhibit transcriptional responses directed by constitutive expression of active interferon regulatory factor IRF3-5D or after stimulation by alpha interferon (IFN-α) or tumor necrosis factor alpha (TNF-α). Consistent with IFN pathway-specific regulation, the ANDV N protein inhibited TBK1-directed IRF3 phosphorylation (phosphorylation of serine 396 [pS396]) and TBK1 autophosphorylation (pS172). Collectively, these findings indicate that the ANDV N inhibits IFN signaling responses by interfering with TBK1 activation, upstream of IRF3 phosphorylation and NF-κB activation. Moreover, our findings reveal that ANDV uniquely carries a gene encoding a virulence determinant within its N protein that is capable of restricting ISG and IFN-β induction and provide a rationale for the novel pathogenesis and spread of ANDV.

IMPORTANCE
Andes virus (ANDV) is distinguished from other hantaviruses by its unique ability to spread from person to person and cause lethal hantavirus pulmonary syndrome (HPS)-like disease in Syrian hamsters. However, virulence determinants that distinguish ANDV from other pathogenic hantaviruses have yet to be defined. Here we reveal that ANDV uniquely contains a virulence determinant within its nucleocapsid (N) protein that potently inhibits innate cellular signaling pathways. This novel function of the N protein provides a new mechanism for hantaviruses to regulate interferon (IFN) and IFN-stimulated gene (ISG) induction that is likely to contribute to the enhanced ability of ANDV to replicate, spread, and cause disease. These findings differentiate ANDV from other HPS-causing hantaviruses and provide a potential target for viral attenuation that needs to be considered in vaccine development.