bluuebunny/crossref_metadata_2025 huggingface dataset:
The Alarmin Interleukin-33 Drives Protective Antiviral CD8
+
T Cell Responses
Authors: Bonilla Weldy V. , Fröhlich Anja , Senn Karin , Kallert Sandra , Fernandez Marylise , Johnson Susan , Kreutzfeldt Mario , Hegazy Ahmed N. , Schrick Christina , Fallon Padraic G. , Klemenz Roman , Nakae Susumu , Adler Heiko , Merkler Doron , Löhning Max , Pinschewer Daniel D.
Published: February 2012

Sound the Alarm

When small protein fragments or nucleic acids derived from an invading pathogen are detected by pattern recognition receptors on immune cells, the innate immune response is triggered. This event activates cells of the adaptive immune system, and together, both responses clear the infection. Infections also induce the release of “danger-associated molecular patterns,” or alarmins, from the host as a result of tissue damage. Whether these are also important for the ensuing immune response is less clear.

Bonilla
et al.

(p.
984
, published online 2 February) report that the alarmin, interleukin-33, is required for optimal cytotoxic CD8
+
T cells responses and antiviral immunity in mice. In virus-infected mice deficient in IL-33 or its receptor, IL-33 is essential for signaling CD8
+
T cells to expand, produce multiple cytokines and acquire cytotoxic capabilities. These results showed that endogenous material, independently of pathogen-derived molecules, are also required for antiviral immunity.
Plant UVR8 Photoreceptor Senses UV-B by Tryptophan-Mediated Disruption of Cross-Dimer Salt Bridges
Authors: Christie John M. , Arvai Andrew S. , Baxter Katherine J. , Heilmann Monika , Pratt Ashley J. , O’Hara Andrew , Kelly Sharon M. , Hothorn Michael , Smith Brian O. , Hitomi Kenichi , Jenkins Gareth I. , Getzoff Elizabeth D.
Published: February 2012

Donuts Dissociate

In
Arabidopsis
, the UVR8 protein responds to ultraviolet-B (UV-B) light by dissociating into monomers, which are then available to interact with downstream factors that enact the plant's response to light.

Christie
et al.

(p.
1492
, published online 9 February; see the cover and see the Perspective by

Gardner and Correa

) have now determined the crystal structure of UVR8. Without ultraviolet-B light, UVR8 dimerizes, with two donut-shaped monomers joined by a network of salt bridges. Close-packing of a pyramid of tryptophan residues permits exciton coupling that is key to UV-B perception. Electron transfer after UV-B perception could dissociate the salt bridges that hold the dimer together and release monomeric UVR8 to initiate light-induced signaling.
Maintaining Equilibrium
Authors: Vinson Valda
Published: February 2012

The arrangement of four central ion-binding sites with differing specificities suggests how a membrane transporter protein works.
Have You Heard About Levitra?
Authors: Wong Wei
Published: February 2012

The type 5 phosphodiesterase inhibitor Levitra (vardenafil) prevents noise-induced hearing loss in rats.
Novel Modeling Framework To Guide Design of Optimal Dosing Strategies for β-Lactamase Inhibitors
Authors: Bhagunde Pratik , Chang Kai-Tai , Hirsch Elizabeth B. , Ledesma Kimberly R. , Nikolaou Michael , Tam Vincent H.
Published: February 2012

The scarcity of new antibiotics against drug-resistant bacteria has led to the development of inhibitors targeting specific resistance mechanisms, which aim to restore the effectiveness of existing agents. However, there are few guidelines for the optimal dosing of inhibitors. Extending the utility of mathematical modeling, which has been used as a decision support tool for antibiotic dosing regimen design, we developed a novel mathematical modeling framework to guide optimal dosing strategies for a beta-lactamase inhibitor. To illustrate our approach, MK-7655 was used in combination with imipenem against a clinical isolate of
Klebsiella pneumoniae
known to produce KPC-2. A theoretical concept capturing fluctuating susceptibility over time was used to define a novel pharmacodynamic index (time above instantaneous MIC [
T
>MIC
i
]). The MK-7655 concentration-dependent MIC reduction was characterized by using a modified sigmoid maximum effect (
E
max
)-type model. Various dosing regimens of MK-7655 were simulated to achieve escalating
T
>MIC
i
values in the presence of a clinical dose of imipenem (500 mg every 6 h). The effectiveness of these dosing exposures was subsequently validated by using a hollow-fiber infection model (HFIM). An apparent trend in the bacterial response was observed in the HFIM with increasing
T
>MIC
i
values. In addition, different dosing regimens of MK-7655 achieving a similar
T
>MIC
i
(69%) resulted in comparable bacterial killing over 48 h. The proposed framework was reasonable in predicting the
in vitro
activity of a novel beta-lactamase inhibitor, and its utility warrants further investigations.
Removal and Transfer of Viruses on Food Contact Surfaces by Cleaning Cloths
Authors: Gibson Kristen E. , Crandall Philip G. , Ricke Steven C.
Published: February 2012

Contamination of food contact surfaces with pathogens is considered an important vehicle for the indirect transmission of food-borne diseases. Five different cleaning cloths were assessed for the ability to remove viruses from food contact surfaces (stainless steel surface and nonporous solid surface) and to transfer viruses back to these surfaces. Cleaning cloths evaluated include two different cellulose/cotton cloths, one microfiber cloth, one nonwoven cloth, and one cotton terry bar towel. Four viral surrogates (murine norovirus [MNV], feline calicivirus [FCV], bacteriophages PRD1 and MS2) were included. Removal of FCV from stainless steel was significantly greater (
P
≤ 0.05) than that from nonporous solid surface, and overall removal of MNV from both surfaces was significantly less (
P
≤ 0.05) than that of FCV and PRD1. Additionally, the terry towel removed significantly fewer total viruses (
P
≤ 0.05) than the microfiber and one of the cotton/cellulose cloths. The cleaning cloth experiments were repeated with human norovirus. For transfer of viruses from cloth to surface, both cellulose/cotton cloths and microfiber transferred an average of 3.4 and 8.5 total PFU, respectively, to both surfaces, and the amounts transferred were significantly different (
P
≤ 0.05) from those for the nonwoven cloth and terry towel (309 and 331 total PFU, respectively). There was no statistically significant difference (
P
> 0.05) in the amount of virus transfer between surfaces. These data indicate that while the cleaning cloths assessed here can remove viruses from surfaces, some cloths may also transfer a significant amount of viruses back to food contact surfaces.
Glyoxylate Metabolism Is a Key Feature of the Metabolic Degradation of 1,4-Dioxane by Pseudonocardia dioxanivorans Strain CB1190
Authors: Grostern Ariel , Sales Christopher M. , Zhuang Wei-Qin , Erbilgin Onur , Alvarez-Cohen Lisa
Published: February 2012

The groundwater contaminant 1,4-dioxane (dioxane) is transformed by several monooxygenase-expressing microorganisms, but only a few of these, includingPseudonocardia dioxanivoransstrain CB1190, can metabolize the compound as a sole carbon and energy source. However, nothing is yet known about the genetic basis of dioxane metabolism. In this study, we used a microarray to study differential expression of genes in strain CB1190 grown on dioxane, glycolate (a previously identified intermediate of dioxane degradation), or pyruvate. Of eight multicomponent monooxygenase gene clusters carried by the strain CB1190 genome, only the monooxygenase gene cluster located on plasmid pPSED02 was upregulated with dioxane relative to pyruvate. Plasmid-borne genes for putative aldehyde dehydrogenases, an aldehyde reductase, and an alcohol oxidoreductase were also induced during growth with dioxane. With both dioxane and glycolate, a chromosomal gene cluster encoding a putative glycolate oxidase was upregulated, as were chromosomal genes related to glyoxylate metabolism through the glyoxylate carboligase pathway. Glyoxylate carboligase activity in cell extracts from cells pregrown with dioxane and inRhodococcus jostiistrain RHA1 cells expressing the putative strain CB1190 glyoxylate carboligase gene further demonstrated the role of glyoxylate metabolism in the degradation of dioxane. Finally, we used13C-labeled dioxane amino acid isotopomer analysis to provide additional evidence that metabolites of dioxane enter central metabolism as three-carbon compounds, likely as phosphoglycerate. The routing of dioxane metabolites via the glyoxylate carboligase pathway helps to explain how dioxane is metabolized as a sole carbon and energy source for strain CB1190.
Microbial Functional Gene Diversity with a Shift of Subsurface Redox Conditions during
In Situ
Uranium Reduction
Authors: Liang Yuting , Van Nostrand Joy D. , N′Guessan Lucie A. , Peacock Aaron D. , Deng Ye , Long Philip E. , Resch C. Tom , Wu Liyou , He Zhili , Li Guanghe , Hazen Terry C. , Lovley Derek R. , Zhou Jizhong
Published: February 2012

To better understand the microbial functional diversity changes with subsurface redox conditions during
in situ
uranium bioremediation, key functional genes were studied with GeoChip, a comprehensive functional gene microarray, in field experiments at a uranium mill tailings remedial action (UMTRA) site (Rifle, CO). The results indicated that functional microbial communities altered with a shift in the dominant metabolic process, as documented by hierarchical cluster and ordination analyses of all detected functional genes. The abundance of
dsrAB
genes (dissimilatory sulfite reductase genes) and methane generation-related
mcr
genes (methyl coenzyme M reductase coding genes) increased when redox conditions shifted from Fe-reducing to sulfate-reducing conditions. The cytochrome genes detected were primarily from
Geobacter
sp. and decreased with lower subsurface redox conditions. Statistical analysis of environmental parameters and functional genes indicated that acetate, U(VI), and redox potential (E
h
) were the most significant geochemical variables linked to microbial functional gene structures, and changes in microbial functional diversity were strongly related to the dominant terminal electron-accepting process following acetate addition. The study indicates that the microbial functional genes clearly reflect the
in situ
redox conditions and the dominant microbial processes, which in turn influence uranium bioreduction. Microbial functional genes thus could be very useful for tracking microbial community structure and dynamics during bioremediation.
Prevalence, Enumeration, Serotypes, and Antimicrobial Resistance Phenotypes of Salmonella enterica Isolates from Carcasses at Two Large United States Pork Processing Plants
Authors: Schmidt John W. , Brichta-Harhay Dayna M. , Kalchayanand Norasak , Bosilevac Joseph M. , Shackelford Steven D. , Wheeler Tommy L. , Koohmaraie Mohammad
Published: February 2012

The objective of this study was to characterize
Salmonella enterica
contamination on carcasses in two large U.S. commercial pork processing plants. The carcasses were sampled at three points, before scalding (prescald), after dehairing/polishing but before evisceration (preevisceration), and after chilling (chilled final). The overall prevalences of
Salmonella
on carcasses at these three sampling points, prescald, preevisceration, and after chilling, were 91.2%, 19.1%, and 3.7%, respectively. At one of the two plants, the prevalence of
Salmonella
was significantly higher (
P
< 0.01) for each of the carcass sampling points. The prevalences of carcasses with enumerable
Salmonella
at prescald, preevisceration, and after chilling were 37.7%, 4.8%, and 0.6%, respectively. A total of 294 prescald carcasses had
Salmonella
loads of >1.9 log CFU/100 cm
2
, but these carcasses were not equally distributed between the two plants, as 234 occurred at the plant with higher
Salmonella
prevalences. Forty-one serotypes were identified on prescald carcasses with
Salmonella enterica
serotypes Derby, Typhimurium, and Anatum predominating.
S. enterica
serotypes Typhimurium and London were the most common of the 24 serotypes isolated from preevisceration carcasses. The
Salmonella
serotypes Johannesburg and Typhimurium were the most frequently isolated serotypes of the 9 serotypes identified from chilled final carcasses. Antimicrobial susceptibility was determined for selected isolates from each carcass sampling point. Multiple drug resistance (MDR), defined as resistance to three or more classes of antimicrobial agents, was identified for 71.2%, 47.8%, and 77.5% of the tested isolates from prescald, preevisceration, and chilled final carcasses, respectively. The results of this study indicate that the interventions used by pork processing plants greatly reduce the prevalence of
Salmonella
on carcasses, but MDR
Salmonella
was isolated from 3.2% of the final carcasses sampled.
Presence and Functionality of Mating Type Genes in the Supposedly Asexual Filamentous Fungus Aspergillus oryzae
Authors: Wada Ryuta , Maruyama Jun-ichi , Yamaguchi Haruka , Yamamoto Nanase , Wagu Yutaka , Paoletti Mathieu , Archer David B. , Dyer Paul S. , Kitamoto Katsuhiko
Published: February 2012

The potential for sexual reproduction in
Aspergillus oryzae
was assessed by investigating the presence and functionality of
MAT
genes. Previous genome studies had identified a
MAT1-1
gene in the reference strain RIB40. We now report the existence of a complementary
MAT1-2
gene and the sequencing of an idiomorphic region from
A. oryzae
strain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of the
MAT1-1
and
MAT1-2
genotypes among 180 strains assayed, including industrial
tane-koji
isolates. Strains used for sake and miso production showed a near-1:1 ratio of the MAT1-1 and MAT1-2 mating types, whereas strains used for soy sauce production showed a significant bias toward the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and gene expression was compared by DNA microarray and quantitative real-time PCR (qRT-PCR) methodologies under conditions in which
MAT
genes were expressed. Thirty-three genes were found to be upregulated more than 10-fold in either the MAT1-1 host strain or the MAT1-2 gene replacement strain relative to each other, showing that both the
MAT1-1
and
MAT1-2
genes functionally regulate gene expression in
A. oryzae
in a mating type-dependent manner, the first such report for a supposedly asexual fungus.
MAT1-1
expression specifically upregulated an α-pheromone precursor gene, but the functions of most of the genes affected were unknown. The results are consistent with a heterothallic breeding system in
A. oryzae
, and prospects for the discovery of a sexual cycle are discussed.
Oxidative Stress Tolerance, Adenylate Cyclase, and Autophagy Are Key Players in the Chronological Life Span of Saccharomyces cerevisiae during Winemaking
Authors: Orozco Helena , Matallana Emilia , Aranda Agustín
Published: February 2012

Most grape juice fermentation takes place when yeast cells are in a nondividing state called the stationary phase. Under such circumstances, we aimed to identify the genetic determinants controlling longevity, known as the chronological life span. We identified commercial strains with both short (EC1118) and long (CSM) life spans in laboratory growth medium and compared them under diverse conditions. Strain CSM shows better tolerance to stresses, including oxidative stress, in the stationary phase. This is reflected during winemaking, when this strain has an increased maximum life span. Compared to EC1118, CSM overexpresses a mitochondrial rhodanese gene-like gene,
RDL2
, whose deletion leads to increased reactive oxygen species production at the end of fermentation and a correlative loss of viability at this point. EC1118 shows faster growth and higher expression of glycolytic genes, and this is related to greater PKA activity due to the upregulation of the adenylate cyclase gene. This phenotype has been linked to the presence of a δ element in its promoter, whose removal increases the life span. Finally, EC1118 exhibits a higher level of protein degradation by autophagy, which might help achieve fast growth at the expense of cellular structures and may be relevant for long-term survival under winemaking conditions.
Characterization of Biphenyl Dioxygenase Sequences and Activities Encoded by the Metagenomes of Highly Polychlorobiphenyl-Contaminated Soils
Authors: Standfuß-Gabisch Christine , Al-Halbouni Djamila , Hofer Bernd
Published: February 2012

Total extracted DNA from two heavily polychlorobiphenyl-contaminated soils was analyzed with respect to biphenyl dioxygenase sequences and activities. This was done by PCR amplification and cloning of a DNA segment encoding the active site of the enzyme. The translated sequences obtained fell into three similarity clusters (I to III). Sequence identities were high within but moderate or low between the clusters. Members of clusters I and II showed high sequence similarities with well-known biphenyl dioxygenases. Cluster III showed low (43%) sequence identity with a biphenyl dioxygenase from
Rhodococcus jostii
RHA1. Amplicons from the three clusters were used to reconstitute and express complete biphenyl dioxygenase operons. In most cases, the resulting hybrid dioxygenases were detected in cell extracts of the recombinant hosts. At least 83% of these enzymes were catalytically active. Several amino acid exchanges were identified that critically affected activity. Chlorobiphenyl turnover by the enzymes containing the prototype sequences of clusters I and II was characterized with 10 congeners that were major, minor, or not constituents of the contaminated soils. No direct correlations were observed between on-site concentrations and rates of productive dioxygenations of these chlorobiphenyls. The prototype enzymes displayed markedly different substrate and product ranges. The cluster II dioxygenase possessed a broader substrate spectrum toward the assayed congeners, whereas the cluster I enzyme was superior in the attack of
ortho
-chlorinated aromatic rings. These results demonstrate the feasibility of the applied approach to functionally characterize dioxygenase activities of soil metagenomes via amplification of incomplete genes.
Bacterial Dimethylsulfoniopropionate Degradation Genes in the Oligotrophic North Pacific Subtropical Gyre
Authors: Varaljay Vanessa A. , Gifford Scott M. , Wilson Samuel T. , Sharma Shalabh , Karl David M. , Moran Mary Ann
Published: February 2012

Dimethylsulfoniopropionate (DMSP) is an organic sulfur compound that is rapidly metabolized by marine bacteria either by cleavage to dimethylsulfide (DMS) or demethylation to 3-methiolpropionate. The abundance and diversity of genes encoding bacterial DMS production (
dddP
) and demethylation (
dmdA
) were measured in the North Pacific subtropical gyre (NPSG) between May 2008 and February 2009 at Station ALOHA (22°45′N, 158°00′W) at two depths: 25 m and the deep chlorophyll maximum (DCM; ∼100 m). The highest abundance of
dmdA
genes was in May 2008 at 25 m, with ∼16.5% of cells harboring a gene in one of the eight subclades surveyed, while the highest abundance of
dddP
genes was in July 2008 at 25 m, with ∼2% of cells harboring a gene. The
dmdA
gene pool was consistently dominated by homologs from SAR11 subclades, which was supported by findings in metagenomic data sets derived from Station ALOHA. Expression of the SAR11
dmdA
genes was low, with typical transcript:gene ratios between 1:350 and 1:1,400. The abundance of DMSP genes was statistically different between 25 m and the DCM and correlated with a number of environmental variables, including primary production, photosynthetically active radiation, particulate DMSP, and DMS concentrations. At 25 m,
dddP
abundance was positively correlated with pigments that are diagnostic of diatoms; at the DCM,
dmdA
abundance was positively correlated with temperature. Based on gene abundance, we hypothesize that SAR11 bacterioplankton dominate DMSP cycling in the oligotrophic NPSG, with lesser but consistent involvement of other members of the bacterioplankton community.
Reduction of Salmonella enterica Serovar Enteritidis Colonization in 20-Day-Old Broiler Chickens by the Plant-Derived Compounds
trans
-Cinnamaldehyde and Eugenol
Authors: Kollanoor-Johny Anup , Mattson Tyler , Baskaran Sangeetha Ananda , Amalaradjou Mary Anne , Babapoor Sankhiros , March Benjamin , Valipe Satyender , Darre Michael , Hoagland Thomas , Schreiber David , Khan Mazhar I. , Donoghue Ann , Donoghue Dan , Venkitanarayanan Kumar
Published: February 2012

The efficacies of
trans
-cinnamaldehyde (TC) and eugenol (EG) for reducing
Salmonella enterica
serovar Enteritidis colonization in broiler chickens were investigated. In three experiments for each compound, 1-day-old chicks (
n
= 75/experiment) were randomly assigned to five treatment groups (
n
= 15/treatment group): negative control (-ve
S
. Enteritidis, -ve TC, or EG), compound control (-ve
S
. Enteritidis, +ve 0.75% [vol/wt] TC or 1% [vol/wt] EG), positive control (+ve
S
. Enteritidis, -ve TC, or EG), low-dose treatment (+ve
S
. Enteritidis, +ve 0.5% TC, or 0.75% EG), and high-dose treatment (+ve
S
. Enteritidis, +ve 0.75% TC, or 1% EG). On day 0, birds were tested for the presence of any inherent
Salmonella
(
n
= 5/experiment). On day 8, birds were inoculated with ∼8.0 log
10
CFU
S
. Enteritidis, and cecal colonization by
S
. Enteritidis was ascertained (
n
= 10 chicks/experiment) after 24 h (day 9). Six birds from each treatment group were euthanized on days 7 and 10 after inoculation, and cecal
S
. Enteritidis numbers were determined. TC at 0.5 or 0.75% and EG at 0.75 or 1% consistently reduced (
P
< 0.05)
S
. Enteritidis in the cecum (≥3 log
10
CFU/g) after 10 days of infection in all experiments. Feed intake and body weight were not different for TC treatments (
P
> 0.05); however, EG supplementation led to significantly lower (
P
< 0.05) body weights. Follow-up
in vitro
experiments revealed that the subinhibitory concentrations (SICs, the concentrations that did not inhibit
Salmonella
growth) of TC and EG reduced the motility and invasive abilities of
S
. Enteritidis and downregulated expression of the motility genes
flhC
and
motA
and invasion genes
hilA
,
hilD
, and
invF
. The results suggest that supplementation with TC and EG through feed can reduce
S
. Enteritidis colonization in chickens.
Influence of Air Quality on the Composition of Microbial Pathogens in Fresh Rainwater
Authors: Kaushik Rajni , Balasubramanian Rajasekhar , de la Cruz Armah A.
Published: February 2012

In this study, the microbiological quality of fresh rainwater was assessed from 50 rain events under tropical weather conditions for a year. The levels of four major opportunistic waterborne pathogens, namely,
Escherichia coli
,
Klebsiella pneumoniae
,
Pseudomonas aeruginosa
, and
Aeromonas hydrophila
, in rainwater samples were quantified by using a robust and sensitive quantitative PCR (qPCR) method. Of the 50 rainwater samples, 25 were found to be positive for at least one pathogen: 21 for
E. coli
, 16 for
P. aeruginosa
, 6 for
K. pneumoniae
, and 1 for
A. hydrophila
. In addition to the microbiological assessment of rainwater samples, we also studied the influence of prevailing air quality on the microbial quality of rainwater over the sampling period. A significant change in the diversity and relative abundance of the basic microbial indicator organisms in rainwater was observed during a major regional air pollution episode in Southeast Asia due to biomass-burning emissions.
The Bacterial Community in the Gut of the Cockroach Shelfordella lateralis Reflects the Close Evolutionary Relatedness of Cockroaches and Termites
Authors: Schauer Christine , Thompson Claire L. , Brune Andreas
Published: February 2012

Termites and cockroaches are closely related, with molecular phylogenetic analyses even placing termites within the radiation of cockroaches. The intestinal tract of wood-feeding termites harbors a remarkably diverse microbial community that is essential for the digestion of lignocellulose. However, surprisingly little is known about the gut microbiota of their closest relatives, the omnivorous cockroaches. Here, we present a combined characterization of physiological parameters, metabolic activities, and bacterial microbiota in the gut of
Shelfordella lateralis
, a representative of the cockroach family Blattidae, the sister group of termites. We compared the bacterial communities within each gut compartment using terminal-restriction fragment length polymorphism (T-RFLP) analysis and made a 16S rRNA gene clone library of the microbiota in the colon—the dilated part of the hindgut with the highest density and diversity of bacteria. The colonic community was dominated by members of the
Bacteroidetes
,
Firmicutes
(mainly
Clostridia
), and some
Deltaproteobacteria. Spirochaetes
and
Fibrobacteres
, which are abundant members of termite gut communities, were conspicuously absent. Nevertheless, detailed phylogenetic analysis revealed that many of the clones from the cockroach colon clustered with sequences previously obtained from the termite gut, which indicated that the composition of the bacterial community reflects at least in part the phylogeny of the host.
New Complementation Constructs for Inducible and Constitutive Gene Expression in Neisseria gonorrhoeae and Neisseria meningitidis
Authors: Ramsey Meghan E. , Hackett Kathleen T. , Kotha Chaitra , Dillard Joseph P.
Published: February 2012

We have created new complementation constructs for use in
Neisseria gonorrhoeae
and
Neisseria meningitidis
. The constructs contain regions of homology with the chromosome and direct the insertion of a gene of interest into the intergenic region between the genes
iga
and
trpB
. In order to increase the available options for gene expression in
Neisseria
, we designed the constructs to contain one of three different promoters. One of the constructs contains the isopropyl-β-
d
-thiogalactopyranoside-inducible
lac
promoter, which has been widely used in
Neisseria
. We also designed a construct that contains the strong, constitutive promoter from the gonococcal
opaB
gene. The third construct contains a tetracycline-inducible promoter, a novel use of this promoter in
Neisseria
. We demonstrate that anhydrotetracycline can be used to induce gene expression in the pathogenic
Neisseria
at very low concentrations and without negatively affecting the growth of the organisms. We use these constructs to complement an arginine auxotrophy in
N. gonorrhoeae
as well as to express a translational fusion of alkaline phosphatase with TraW. TraW is a component of the gonococcal type IV secretion system, and we demonstrate that TraW localizes to the periplasm.
Effects of Sporulation Conditions on the Germination and Germination Protein Levels of Bacillus subtilis Spores
Authors: Ramirez-Peralta Arturo , Zhang Pengfei , Li Yong-qing , Setlow Peter
Published: February 2012

Bacillus subtilis
spores prepared in rich medium germinated faster with nutrient germinants than poor-medium spores as populations in liquid and multiple individual spores on a microscope slide. Poor-medium spores had longer average lag times between mixing of spores with nutrient germinants and initiation of Ca-dipicolinic acid (CaDPA) release. Rich-medium spores made at 37°C germinated slightly faster with nutrient germinants than 23°C spores in liquid, but not when spores germinated on a slide. The difference in germination characteristics of these spore populations in liquid was paralleled by changes in expression levels of a transcriptional
lacZ
fusion to the
gerA
operon, encoding a germinant receptor (GR). Levels of GR subunits were 3- to 8-fold lower in poor-medium spores than rich-medium spores and 1.6- to 2-fold lower in 23°C spores than 37°C spores, and levels of the auxiliary germination protein GerD were 3.5- to 4-fold lower in poor medium and 23°C spores. In contrast, levels of another likely germination protein, SpoVAD, were similar in all these spores. These different spores germinated similarly with CaDPA, and poor-medium and 23°C spores germinated faster than rich-medium and 37°C spores, respectively, with dodecylamine. Since spore germination with CaDPA and dodecylamine does not require GerD or GRs, these results indicate that determinants of rates of nutrient germination of spores prepared differently are primarily the levels of the GRs that bind nutrient germinants and trigger germination and secondarily the levels of GerD.
Wsp1 Is Downstream of Cin1 and Regulates Vesicle Transport and Actin Cytoskeleton as an Effector of Cdc42 and Rac1 in Cryptococcus neoformans
Authors: Shen Gui , Zhou Erxun , Alspaugh J. Andrew , Wang Ping
Published: February 2012

Human Wiskott-Aldrich syndrome protein (WASP) is a scaffold linking upstream signals to the actin cytoskeleton. In response to intersectin ITSN1 and Rho GTPase Cdc42, WASP activates the Arp2/3 complex to promote actin polymerization. The human pathogen
Cryptococcus neoformans
contains the ITSN1 homolog Cin1 and the WASP homolog Wsp1, which share more homology with human proteins than those of other fungi. Here we demonstrate that Cin1, Cdc42/Rac1, and Wsp1 function in an effector pathway similar to that of mammalian models. In the
cin1
mutant, expression of the autoactivated Wsp1-B-GBD allele partially suppressed the mutant defect in endocytosis, and expression of the constitutively active
CDC42

Q61L

allele restored normal actin cytoskeleton structures. Similar phenotypic suppression can be obtained by the expression of a Cdc42-green fluorescent protein (GFP)-Wsp1 fusion protein. In addition, Rac1, which was found to exhibit a role in early endocytosis, activates Wsp1 to regulate vacuole fusion. Rac1 interacted with Wsp1 and depended on Wsp1 for its vacuolar membrane localization. Expression of the Wsp1-B-GBD allele restored vacuolar membrane fusion in the
rac1
mutant. Collectively, our studies suggest novel ways in which this pathogenic fungus has adapted conserved signaling pathways to control vesicle transport and actin organization, likely benefiting survival within infected hosts.
Morphology of the Trypanosome Bilobe, a Novel Cytoskeletal Structure
Authors: Esson Heather J. , Morriswood Brooke , Yavuz Sevil , Vidilaseris Keni , Dong Gang , Warren Graham
Published: February 2012

The trypanosome bilobe is a cytoskeletal structure of unclear function. To date, four proteins have been shown to localize stably to it: TbMORN1, TbLRRP1, TbCentrin2, and TbCentrin4. In this study, a combination of immunofluorescence microscopy and electron microscopy was used to explore the morphology of the bilobe and its relationship to other nearby cytoskeletal structures in the African trypanosome procyclic trypomastigote. The use of detergent/salt-extracted flagellum preparations was found to be an effective way of discerning features of the cytoskeletal ultrastructure that are normally obscured. TbMORN1 and TbCentrin4 together define a hairpin structure comprising an arm of TbCentrin4 and a fishhook of TbMORN1. The two arms flank a specialized microtubule quartet and the flagellum attachment zone filament, with TbMORN1 running alongside the former and TbCentrin4 alongside the latter. The hooked part of TbMORN1 sits atop the flagellar pocket collar marked by TbBILBO1. The TbMORN1 bilobe occasionally exhibits tendrillar extensions that seem to be connected to the basal and probasal bodies. The TbMORN1 molecules present on these tendrils undergo higher rates of turnover than those for molecules on the main bilobe structure. These observations have been integrated with previous detailed descriptions of the cytoskeletal elements in trypanosome cells.
Role of a SpoVA Protein in Dipicolinic Acid Uptake into Developing Spores of Bacillus subtilis
Authors: Li Yunfeng , Davis Andrew , Korza George , Zhang Pengfei , Li Yong-qing , Setlow Barbara , Setlow Peter , Hao Bing
Published: February 2012

The proteins encoded by thespoVAoperon, including SpoVAD, are essential for the uptake of the 1:1 chelate of pyridine-2,6-dicarboxylic acid (DPA2,6) and Ca2+into developing spores of the bacteriumBacillus subtilis. The crystal structure ofB. subtilisSpoVAD has been determined recently, and a structural homology search revealed that SpoVAD shares significant structural similarity but not sequence homology to a group of enzymes that bind to and/or act on small aromatic molecules. We find that molecular docking placed DPA2,6exclusively in a highly conserved potential substrate-binding pocket in SpoVAD that is similar to that in the structurally homologous enzymes. We further demonstrate that SpoVAD binds both DPA2,6and Ca2+-DPA2,6with a similar affinity, while exhibiting markedly weaker binding to other DPA isomers. Importantly, mutations of conserved amino acid residues in the putative DPA2,6-binding pocket in SpoVAD essentially abolish its DPA2,6-binding capacity. Moreover, replacement of the wild-typespoVADgene inB. subtiliswith any of thesespoVADgene variants effectively eliminated DPA2,6uptake into developing spores in sporulation, although the variant proteins were still located in the spore inner membrane. Our results provide direct evidence that SpoVA proteins, in particular SpoVAD, are directly involved in DPA2,6movement into developingB. subtilisspores.
The Yersinia pestis Rcs Phosphorelay Inhibits Biofilm Formation by Repressing Transcription of the Diguanylate Cyclase Gene
hmsT
Authors: Sun Yi-Cheng , Guo Xiao-Peng , Hinnebusch B. Joseph , Darby Creg
Published: February 2012

Yersinia pestis
, which causes bubonic plague, forms biofilms in fleas, its insect vectors, as a means to enhance transmission. Biofilm development is positively regulated by
hmsT
, encoding a diguanylate cyclase that synthesizes the bacterial second messenger cyclic-di-GMP. Biofilm development is negatively regulated by the Rcs phosphorelay signal transduction system. In this study, we show that Rcs-negative regulation is accomplished by repressing transcription of
hmsT.
Effect of Periodontal Pathogens on the Metatranscriptome of a Healthy Multispecies Biofilm Model
Authors: Frias-Lopez Jorge , Duran-Pinedo Ana
Published: February 2012

Oral bacterial biofilms are highly complex microbial communities with up to 700 different bacterial taxa. We report here the use of metatranscriptomic analysis to study patterns of community gene expression in a multispecies biofilm model composed of species found in healthy oral biofilms (
Actinomyces naeslundii
,
Lactobacillus casei
,
Streptococcus mitis
,
Veillonella parvula
, and
Fusobacterium nucleatum
) and the same biofilm plus the periodontopathogens
Porphyromonas gingivalis
and
Aggregatibacter actinomycetemcomitans
. The presence of the periodontopathogens altered patterns in gene expression, and data indicate that transcription of protein-encoding genes and small noncoding RNAs is stimulated. In the healthy biofilm hypothetical proteins, transporters and transcriptional regulators were upregulated while chaperones and cell division proteins were downregulated. However, when the pathogens were present, chaperones were highly upregulated, probably due to increased levels of stress. We also observed a significant upregulation of ABC transport systems and putative transposases. Changes in Clusters of Orthologous Groups functional categories as well as gene set enrichment analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed that in the absence of pathogens, only sets of proteins related to transport and secondary metabolism were upregulated, while in the presence of pathogens, proteins related to growth and division as well as a large portion of transcription factors were upregulated. Finally, we identified several small noncoding RNAs whose predicted targets were genes differentially expressed in the open reading frame libraries. These results show the importance of pathogens controlling gene expression of a healthy oral community and the usefulness of metatranscriptomic techniques to study gene expression profiles in complex microbial community models.
Biochemical Characterization of the CDP-
d
-Arabinitol Biosynthetic Pathway in Streptococcus pneumoniae 17F
Authors: Wang Quan , Xu Yanli , Perepelov Andrei V. , Knirel Yuriy A. , Reeves Peter R. , Shashkov Alexander S. , Guo Xi , Ding Peng , Feng Lu
Published: February 2012

Streptococcus pneumoniae
is a major human pathogen associated with many diseases worldwide. Capsular polysaccharides (CPSs) are the major virulence factor. The biosynthetic pathway of
d
-arabinitol, which is present in the CPSs of several
S. pneumoniae
serotypes, has never been identified. In this study, the genes
abpA
(previously known as
abp1
) and
abpB
(previously known as
abp2
), which have previously been reported to be responsible for nucleoside diphosphate (NDP)-
d
-arabinitol (the nucleotide-activated form of
d
-arabinitol) synthesis, were cloned. The enzyme products were overexpressed, purified, and analyzed for their respective activities. Novel products produced by AbpA- and AbpB-catalyzing reactions were detected by capillary electrophoresis, and the structures of the products were elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. As a result,
abpA
was identified to be a
d
-xylulose-5-phosphate cytidylyltransferase-encoding gene, responsible for the transfer of CTP to
d
-xylulose-5-phosphate (
d
-Xlu-5-P) to form CDP-
d
-xylulose, and
abpB
was characterized to be a CDP-
d
-xylulose reductase-encoding gene, responsible for the conversion of CDP-
d
-xylulose to CDP-
d
-arabinitol as the final product. The kinetic parameters of AbpA for the substrates
d
-Xlu-5-P and CTP and those of AbpB for the substrate CDP-
d
-xylulose and the cofactors NADH or NADPH were measured, and the effects of temperature, pH, and cations on the two enzymes were analyzed. This study confirmed the involvement of the genes
abpA
and
abpB
and their products in the biosynthetic pathway of CDP-
d
-arabinitol.
Biological Roles of Nontypeable Haemophilus influenzae Type IV Pilus Proteins Encoded by the
pil
and
com
Operons
Authors: Carruthers Michael D. , Tracy Erin N. , Dickson Amanda C. , Ganser Kara B. , Munson Robert S. , Bakaletz Lauren O.
Published: February 2012

We previously demonstrated that one or more products of the genes in the
pil
and
com
gene clusters of the opportunistic human respiratory pathogen nontypeable
Haemophilus influenzae
(NTHI) are required for type IV pilus (Tfp) biogenesis and function. Here, we have now demonstrated that the
pilABCD
and
comABCDEF
gene clusters are operons and that the product of each gene is essential for normal pilus function. Mutants with nonpolar deletions in each of the 10
pil
and
com
genes had an adherence defect when primary human airway cells were used as the target. These mutants were also diminished in their ability to form a biofilm
in vitro
and, additionally, were deficient in natural transformation. Collectively, our data demonstrate that the product of each gene within these operons is required for the normal biogenesis and/or function of NTHI Tfp. Based on the similarity of PilA to other type IV pilins, we further predicted that the product of the
pilA
gene would be the major pilin subunit. Toward that end, we also demonstrated by immunogold labeling and mass spectrometry that PilA is indeed the majority type IV pilin protein expressed by NTHI. These new observations set the stage for experiments designed to dissect the function of each of the proteins encoded by genes within the
pil
and
com
gene clusters. The ability to characterize individual proteins with vital roles in NTHI colonization or pathogenesis has the potential to reduce the burden of NTHI-induced diseases through development of a Tfp-derived vaccine or a pilus-directed therapeutic.
Spontaneous Excision of the O-Polysaccharide
wbkA
Glycosyltranferase Gene Is a Cause of Dissociation of Smooth to Rough Brucella Colonies
Authors: Mancilla Marcos , Marín Clara M. , Blasco José M. , Zárraga Ana María , López-Goñi Ignacio , Moriyón Ignacio
Published: February 2012

The brucellae are Gram-negative pathogens that cause brucellosis, a zoonosis of worldwide importance. The genus
Brucella
includes smooth and rough species that differ in that they carry smooth and rough lipopolysaccharides, respectively.
Brucella abortus
,
B. melitensis
, and
B. suis
are typical smooth species. However, these smooth brucellae dissociate into rough mutants devoid of the lipopolysaccharide O-polysaccharide, a major antigen and a virulence determinant encoded in regions
wbo
(included in genomic island-2) and
wbk
. We demonstrate here the occurrence of spontaneous recombination events in those three
Brucella
species leading to the deletion of a 5.5-kb fragment carrying the
wbkA
glycosyltranferase gene and to the appearance of rough mutants. Analysis of the recombination intermediates suggested homologous recombination between the IS
Bm1
insertion sequences flanking
wbkA
as the mechanism generating the deletion. Excision of
wbkA
was reduced but not abrogated in a
recA
-deficient mutant, showing the existence of both RecA-dependent and -independent processes. Although the involvement of the IS
Bm1
copies flanking
wbkA
suggested a transpositional event, the predicted transpositional joint could not be detected. This absence of detectable transposition was consistent with the presence of polymorphism in the inverted repeats of one of the IS
Bm1
copies. The spontaneous excision of
wbkA
represents a novel dissociation mechanism of smooth brucellae that adds to the previously described excision of genomic island-2. This IS
Bm1
-mediated
wbkA
excision and the different %GC levels of the excised fragment and of other
wbk
genes suggest that the
Brucella
wbk
locus is the result of at least two horizontal acquisition events.
Hfq Regulates Biofilm Gut Blockage That Facilitates Flea-Borne Transmission of Yersinia pestis
Authors: Rempe Katherine A. , Hinz Angela K. , Vadyvaloo Viveka
Published: February 2012

The plague bacillus
Yersinia pestis
can achieve transmission by biofilm blockage of the foregut proventriculus of its flea vector. Hfq is revealed to be essential for biofilm blockage formation and acquisition and fitness of
Y. pestis
during flea gut infection, consistent with posttranscriptional regulatory mechanisms in plague transmission.
Second-Site Suppression of RNase E Essentiality by Mutation of thedeaDRNA Helicase in Escherichia coli
Authors: Tamura Masaru , Kers Johan A. , Cohen Stanley N.
Published: February 2012

Escherichia colicells normally require RNase E activity to propagate and form colonies. Using random Tn10insertion mutagenesis, we screened for second-site suppressor mutations that restore colony-forming ability (CFA) toE. colicells lacking RNase E function and found mutations in three separate chromosomal loci that had this phenotype. Restoration of CFA by mutations in two of the genes identified was observed only in nutrient-poor medium, whereas the effects of mutation of the ATP-dependent RNA helicase DeaD were medium independent. Suppression of thernemutant phenotype by inactivation ofdeaDwas partial, asrne deaDdoubly mutant bacteria had a greatly prolonged generation time and grew as filamentous chains in liquid medium. Moreover, we found that CFA restoration bydeaDinactivation requires normal expression of the endogenousrnggene in doubly mutantrne deaDcells. Second-site suppression bydeaDmutation was attributable specifically to ablation of the helicase activity of DeaD and was reversed by adventitious expression of RhlE or RNase R, both of which can unwind double-stranded RNA. Our results suggest a previously unsuspected role for RNA secondary structure as a determinant of RNase E essentiality.
Anaerobic
p
-Coumarate Degradation by Rhodopseudomonas palustris and Identification of CouR, a MarR Repressor Protein That Binds
p
-Coumaroyl Coenzyme A
Authors: Hirakawa Hidetada , Schaefer Amy L. , Greenberg E. Peter , Harwood Caroline S.
Published: February 2012

The phenylpropanoid
p
-coumarate and structurally related aromatic compounds are produced in large amounts by green plants and are excellent carbon sources for many soil bacteria. Aerobic bacteria remove the acyl side chain from phenylpropanoids to leave an aromatic aldehyde, which then enters one of several possible central pathways of benzene ring degradation. We investigated the pathway for the anaerobic degradation of
p
-coumarate by the phototrophic bacterium
Rhodopseudomonas palustris
and found that it also follows this metabolic logic. We characterized enzymes for the conversion of
p
-coumarate to
p
-hydroxybenzaldehyde and acetyl coenzyme A (acetyl-CoA) encoded by the
couAB
operon. We also identified a MarR family transcriptional regulator that we named CouR. A
couR
mutant had elevated
couAB
expression. In addition, His-tagged CouR bound with high affinity to a DNA fragment encompassing the
couAB
promoter region, and binding was abrogated by the addition of nanomolar quantities of
p
-coumaroyl-CoA but not by
p
-coumarate. Footprinting demonstrated binding of CouR to an inverted repeat sequence that overlaps the −10 region of the
couAB
promoter. Our results provide evidence for binding of a CoA-modified aromatic compound by a MarR family member. Although the MarR family is widely distributed in bacteria and archaea and includes over 12,000 members, ligands have been identified for relatively few family members. Here we provide biochemical evidence for a new category of MarR ligand.
A primary care Symptoms Clinic for patients with medically unexplained symptoms: pilot randomised trial
Authors: Burton Christopher , Weller David , Marsden Wendy , Worth Allison , Sharpe Michael
Published: February 2012

Objectives

To conduct a pilot trial of a primary care Symptoms Clinic for patients with medically unexplained symptoms and evaluate recruitment and retention, and acceptability of the intervention and to estimate potential treatment effects for a full trial.

Trial design

Randomised parallel group pilot trial.

Setting

Primary care in one locality.

Participants

Primary care database and postal questionnaire were used to identify patients with multiple specialist referrals and multiple physical symptoms unlikely to be explained by disease.

Interventions

General practitioner (GP) with special interest ‘Symptoms Clinic’ + usual care versus usual care alone. The Symptoms Clinic comprised one long (1 h) and three short (20 min) appointments.

Outcomes

Number of patients identified and recruited; acceptability of the intervention (items from Client Satisfaction Questionnaire and interview); Medical Outcomes Survey Short Form 12 (SF-12) physical component summary.

Randomisation

Automated blocked randomisation accessed by telephone.

Blinding

None.

Numbers randomized

16 to intervention and 16 to usual care alone.

Recruitment

72 patients, from seven GP practices, had repeated specialist referrals and a Patient Health Questionnaire (PHQ)-15 score of ≥10 indicating a high probability of medically unexplained symptoms. 15 were ineligible and 25 declined to participate.

Numbers analysed

26 patients; two patients randomised to the intervention group were incorrectly included, three patients in the intervention group and one control did not complete outcome measures.

Outcome

Most patients randomised to the Symptoms Clinic found the intervention acceptable: eight out of 11 reported the intervention helped them to deal with their problems. The mean difference between groups in SF-12 physical component summary, adjusted for baseline, was 3.8 points (SD 6).

Harms

No observed harms.

Conclusions

Patients with multiple medically unexplained symptoms can be systematically identified in primary care; a randomised trial comparing the Symptoms Clinic with usual care is feasible and has the potential to show clinically meaningful benefit.

Trial registration

ISRCTN63083469.