bluuebunny/crossref_metadata_2025 huggingface dataset:
Diet-Induced Metabolic Improvements in a Hamster Model of Hypercholesterolemia Are Strongly Linked to Alterations of the Gut Microbiota
Authors: Martínez Inés , Wallace Grant , Zhang Chaomei , Legge Ryan , Benson Andrew K. , Carr Timothy P. , Moriyama Etsuko N. , Walter Jens
Published: May 2009

The mammalian gastrointestinal microbiota exerts a strong influence on host lipid and cholesterol metabolism. In this study, we have characterized the interplay among diet, gut microbial ecology, and cholesterol metabolism in a hamster model of hypercholesterolemia. Previous work in this model had shown that grain sorghum lipid extract (GSL) included in the diet significantly improved the high-density lipoprotein (HDL)/non-HDL cholesterol equilibrium (T. P. Carr, C. L. Weller, V. L. Schlegel, S. L. Cuppett, D. M. Guderian, Jr., and K. R. Johnson, J. Nutr.
135:
2236-2240, 2005). Molecular analysis of the hamsters' fecal bacterial populations by pyrosequencing of 16S rRNA tags, PCR-denaturing gradient gel electrophoresis, and
Bifidobacterium
-specific quantitative real-time PCR revealed that the improvements in cholesterol homeostasis induced through feeding the hamsters GSL were strongly associated with alterations of the gut microbiota. Bifidobacteria, which significantly increased in abundance in hamsters fed GSL, showed a strong positive association with HDL plasma cholesterol levels (
r
= 0.75;
P
= 0.001). The proportion of members of the family
Coriobacteriaceae
decreased when the hamsters were fed GSL and showed a high positive association with non-HDL plasma cholesterol levels (
r
= 0.84;
P
= 0.0002). These correlations were more significant than those between daily GSL intake and animal metabolic markers, implying that the dietary effects on host cholesterol metabolism are conferred, at least in part, through an effect on the gut microbiota. This study provides evidence that modulation of the gut microbiota-host metabolic interrelationship by dietary intervention has the potential to improve mammalian cholesterol homeostasis, which has relevance for cardiovascular health.
Pyrosequencing of Tagged 16S rRNA Gene Amplicons for Rapid Deciphering of the Microbiomes of Fermented Foods Such as Pearl Millet Slurries
Authors: Humblot Christèle , Guyot Jean-Pierre
Published: May 2009

Pearl millet slurries, mixed with groundnuts or not, were chosen as a model to investigate the feasibility of obtaining a rapid overview of community structure and population dynamics of fermented foods using pyrosequencing of tagged 16S rRNA gene amplicons. From 14 fermented samples collected either in a traditional small-scale processing unit in Burkina Faso or at laboratory scale, 137,469 sequences of bacterial 16S rRNA gene amplicons were characterized. Except for a few
Proteobacteria
, almost all the bacterial sequences were attributed to cultivable bacteria. This approach enabled 80.7% of the sequences to be attributed to a family and 70% to a genus but did not enable identification to the species level. The bacterial sequences were assigned to four phyla, with
Firmicutes
representing the highest diversity, followed by
Proteobacteria
,
Actinobacteria
, and
Bacteroidetes
, which were found only in the slurries prepared in traditional production units. Most of the
Firmicutes
were lactic acid bacteria, mainly represented by members of the
Lactobacillus
,
Pediococcus
,
Leuconostoc
, and
Weissella
genera, whose ratio varied from the onset to the end of the fermentation. The other bacteria present at the beginning of fermentation were generally no longer detected at the end, which is consistent with already-known patterns in the microbial ecology of fermented foods. In conclusion, this method seems very promising for rapid and preliminary microbial characterization in many samples of an unknown food sample, by determining numerous nucleic sequences simultaneously without the need for cloning and cultivation-dependent methods.
Versatile Solidified Nanofibrous Cellulose-Containing Media for Growth of Extremophiles
Authors: Tsudome Mikiko , Deguchi Shigeru , Tsujii Kaoru , Ito Susumu , Horikoshi Koki
Published: May 2009

Solidified media that employ a porous matrix of nanofibrous cellulose are described. The physicochemical stability of the porous structure allows the development of solidified media that can support the growth of extremophiles, such as acidophilic
Acidiphilium
, alkaliphilic
Bacillus
, thermophilic
Geobacillus
and
Thermus
, alkalithermophilic
Bacillus
, and acidothermophilic
Sulfolobus
microbes. The cellulose-supported media have several advantages over agar- and gellan gum-derived media, including versatility and stability.
Screening of Thermotolerant
Gluconobacter
Strains for Production of 5-Keto-
d
-Gluconic Acid and Disruption of Flavin Adenine Dinucleotide-Containing
d
-Gluconate Dehydrogenase
Authors: Saichana Ittipon , Moonmangmee Duangtip , Adachi Osao , Matsushita Kazunobu , Toyama Hirohide
Published: May 2009

We isolated thermotolerant
Gluconobacter
strains that are able to produce 5-keto-
d
-gluconic acid (5KGA) at 37°C, a temperature at which regular mesophilic 5KGA-producing strains showed much less growth and 5KGA production. The thermotolerant strains produced 2KGA as the major product at both 30 and 37°C. The amount of ketogluconates produced at 37°C was slightly less than the amount produced at 30°C. To improve the yield of 5KGA in these strains, we disrupted flavin adenine dinucleotide-gluconate dehydrogenase (FAD-GADH), which is responsible for 2KGA production. Genes for FAD-GADH were cloned by using inverse PCR and an in vitro cloning strategy. The sequences obtained for three thermotolerant strains were identical and showed high levels of identity to the FAD-GADH sequence reported for the genome of
Gluconobacter oxydans
621 H. A kanamycin resistance gene cassette was used to disrupt the FAD-GADH genes in the thermotolerant strains. The mutant strains produced 5KGA exclusively, and the final yields were over 90% at 30°C and 50% at 37°C. We found that the activity of pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase, which is responsible for 5KGA production, increased in response to addition of PQQ and CaCl
2
in vitro when cells were grown at 37°C. Addition of 5 mM CaCl
2
to the culture medium of the mutant strains increased 5KGA production to the point where over 90% of the initial substrate was converted. The thermotolerant
Gluconobacter
strains that we isolated in this study provide a promising new option for industrial 5KGA production.
Phylogeny and Virulence of Naturally Occurring Type III Secretion System-Deficient
Pectobacterium
Strains
Authors: Kim Hye-Sook , Ma Bing , Perna Nicole T. , Charkowski Amy O.
Published: May 2009

Pectobacterium
species are enterobacterial plant-pathogenic bacteria that cause soft rot disease in diverse plant species. Previous epidemiological studies of
Pectobacterium
species have suffered from an inability to identify most isolates to the species or subspecies level. We used three previously described DNA-based methods, 16S-23S intergenic transcribed spacer PCR-restriction fragment length polymorphism analysis, multilocus sequence analysis (MLSA), and pulsed-field gel electrophoresis, to examine isolates from diseased stems and tubers and found that MLSA provided the most reliable classification of isolates. We found that strains belonging to at least two
Pectobacterium
clades were present in each field examined, although representatives of only three of five
Pectobacterium
clades were isolated. Hypersensitive response and DNA hybridization assays revealed that strains of both
Pectobacterium carotovorum
and
Pectobacterium wasabiae
lack a type III secretion system (T3SS). Two of the T3SS-deficient strains assayed lack genes adjacent to the T3SS gene cluster, suggesting that multiple deletions occurred in
Pectobacterium
strains in this locus, and all strains appear to have only six rRNA operons instead of the seven operons typically found in
Pectobacterium
strains. The virulence of most of the T3SS-deficient strains was similar to that of T3SS-encoding strains in stems and tubers.
In Situ Production of Crenarchaeol in Two California Hot Springs
Authors: Pitcher Angela , Schouten Stefan , Sinninghe Damsté Jaap S.
Published: May 2009

Crenarchaeol, a membrane-spanning glycerol dialkyl glycerol tetraether (GDGT) containing a cyclohexane moiety in addition to four cyclopentane moieties, was originally hypothesized to be synthesized exclusively by the mesophilic
Crenarchaeota
. Recent studies reporting the occurrence of crenarchaeol in hot springs and as a membrane constituent of the recently isolated thermophilic crenarchaeote “
Candidatus
Nitrosocaldus yellowstonii,” however, have raised questions regarding its taxonomic distribution and function. To determine whether crenarchaeol in hot springs is indeed synthesized by members of the
Archaea
in situ or is of allochthonous origin, we quantified crenarchaeol present in the form of both intact polar lipids (IPLs) and core lipids in sediments of two California hot springs and in nearby soils. IPL-derived crenarchaeol (IPL-crenarchaeol) was found in both hot springs and soils, suggesting in situ production of this GDGT over a wide temperature range (12°C to 89°C). Quantification of archaeal
amoA
gene abundance by quantitative PCR showed a good correspondence with IPL-crenarchaeol, suggesting that it was indeed derived from living cells and that crenarchaeol-synthesizing members of the
Archaea
in our samples may also be ammonia oxidizers.
Response of
Listeria monocytogenes
to Disinfection Stress at the Single-Cell and Population Levels as Monitored by Intracellular pH Measurements and Viable-Cell Counts
Authors: Kastbjerg Vicky G. , Nielsen Dennis S. , Arneborg Nils , Gram Lone
Published: May 2009

Listeria monocytogenes
has a remarkable ability to survive and persist in food production environments. The purpose of the present study was to determine if cells in a population of
L. monocytogenes
differ in sensitivity to disinfection agents as this could be a factor explaining persistence of the bacterium. In situ analyses of
Listeria monocytogenes
single cells were performed during exposure to different concentrations of the disinfectant Incimaxx DES to study a possible population subdivision. Bacterial survival was quantified with plate counting and disinfection stress at the single-cell level by measuring intracellular pH (pH
i
) over time by fluorescence ratio imaging microscopy. pH
i
values were initially 7 to 7.5 and decreased in both attached and planktonic
L. monocytogenes
cells during exposure to sublethal and lethal concentrations of Incimaxx DES. The response of the bacterial population was homogenous; hence, subpopulations were not detected. However, pregrowth with NaCl protected the planktonic bacterial cells during disinfection with Incimaxx (0.0015%) since pH
i
was higher (6 to 6.5) for the bacterial population pregrown with NaCl than for cells grown without NaCl (pH
i
5 to 5.5) (
P
< 0.05). The protective effect of NaCl was reflected by viable-cell counts at a higher concentration of Incimaxx (0.0031%), where the salt-grown population survived better than the population grown without NaCl (
P
< 0.05). NaCl protected attached cells through drying but not during disinfection. This study indicates that a population of
L. monocytogenes
cells, whether planktonic or attached, is homogenous with respect to sensitivity to an acidic disinfectant studied on the single-cell level. Hence a major subpopulation more tolerant to disinfectants, and hence more persistent, does not appear to be present.
Isolation from Sewage Influent and Characterization of Novel
Staphylococcus aureus
Bacteriophages with Wide Host Ranges and Potent Lytic Capabilities
Authors: Synnott Aidan J. , Kuang Ying , Kurimoto Miki , Yamamichi Keiko , Iwano Hidetomo , Tanji Yasunori
Published: May 2009

Staphylococcus aureus
is a gram-positive pathogen that causes a variety of diseases, including bovine mastitis, which has severe economic consequences. Standard antibiotic treatment results in selection of resistant strains, leading to a need for alternative treatments, such as bacteriophage therapy. Forty-nine
S. aureus
isolates were obtained from the milk of mastitic cows for use in screening of staphylococcal phages. Fifteen isolates which were positive for both coagulase and hemolysin were assayed by PCR for variation in the X region and the immunoglobulin G-binding region of the protein A gene (
spa
) and in the carboxy terminus of the coagulase gene (
coa
) and for the presence of enterotoxin C, G, H, and I genes. The host ranges of 52 phages isolated from sewage influent were determined by performing spot tests with the 15
S. aureus
isolates, and two phages were subsequently chosen for further analysis. ΦSA039 had the widest host range, producing clear plaques on 13 of the 15 isolates (87%), while ΦSA012 produced clear plaques on 8 isolates (53%) and was the only phage that produced a clear plaque on a nonmastitic
S. aureus
strain. Transmission electron microscopy revealed that the phages were similar sizes and belonged to the
Myoviridae
family. Measurement of optical densities during coculture with
S. aureus
isolates confirmed the breadth of the ΦSA039 host range and showed that ΦSA012 had potent lytic capability. ΦSA012-resistant bacteria did not appear for three of seven isolates tested (43%) after 65 h of incubation. These two phages are proposed as candidates for phage therapy of bovine mastitis.
Attenuation ofEdwardsiella tardaVirulence by Small Peptides That Interfere with LuxS/Autoinducer Type 2 Quorum Sensing
Authors: Zhang Min , Jiao Xu-dong , Hu Yong-hua , Sun Li
Published: May 2009

Edwardsiella tardais a gram-negative pathogen with a broad host range that includes humans, animals, and fish. Recent studies have shown that the LuxS/autoinducer type 2 (AI-2) quorum sensing system is involved in the virulence ofE. tarda. In the present study, it was found that theE. tardaLuxS mutants bearing deletions of the catalytic site (C site) and the tyrosine kinase phosphorylation site, respectively, are functionally inactive and that these dysfunctional mutants can interfere with the activity of the wild-type LuxS. Two small peptides, 5411 and 5906, which share sequence identities with the C site of LuxS, were identified. 5411 and 5906 proved to be inhibitors of AI-2 activity and could vitiate the infectivity of the pathogenicE. tardastrain TX1. The inhibitory effect of 5411 and 5906 on AI-2 activity is exerted on LuxS, with which these peptides specifically interact. The expression of 5411 and 5906 in TX1 has multiple effects (altering biofilm production and the expression of certain virulence-associated genes), which are similar to those caused by interruption ofluxSexpression. Further study found that it is very likely that 5411 and 5906 can be released from the strains expressing them and, should TX1 be in the vicinity, captured by TX1. Based on this observation, a constitutive 5411 producer (Pseudomonassp. strain FP3/pT5411) was constructed in the form of a fish commensal isolate that expresses 5411 from a plasmid source. The presence of FP3/pT5411 in fish attenuates the virulence of TX1. Finally, it was demonstrated that fish expressing 5411 directly from tissues exhibit enhanced resistance against TX1 infection.
Influence of Culture Conditions and Medium Composition on the Production of Cellulose by Shiga Toxin-Producing
Escherichia coli
Cells
Authors: Yoo Byong Kwon , Chen Jinru
Published: May 2009

Culture conditions favoring cellulose production by Shiga toxin-producing
Escherichia coli
included a 28°C incubation temperature, an aerobic atmosphere, and the presence of 2% ethanol in Luria-Bertani no-salt agar with pH 6.0 and a water activity of 0.99. These findings will assist in formulating microbiological media useful for cellulose and biofilm research.
Propagation of Human Enteropathogens in Constructed Horizontal Wetlands Used for Tertiary Wastewater Treatment
Authors: Graczyk Thaddeus K. , Lucy Frances E. , Tamang Leena , Mashinski Yessika , Broaders Michael A. , Connolly Michelle , Cheng Hui-Wen A.
Published: May 2009

Constructed subsurface flow (SSF) and free-surface flow (FSF) wetlands are being increasingly implemented worldwide into wastewater treatments in response to the growing need for microbiologically safe reclaimed waters, which is driven by an exponential increase in the human population and limited water resources. Wastewater samples from four SSF and FSF wetlands in northwestern Ireland were tested qualitatively and quantitatively for
Cryptosporidium
spp.,
Giardia duodenalis
, and human-pathogenic microsporidia, with assessment of their viability. Overall, seven species of human enteropathogens were detected in wetland influents, vegetated areas, and effluents:
Cryptosporidium parvum
,
C. hominis
,
C. meleagridis
,
C. muris
,
G. duodenalis
,
Encephalitozoon hellem
, and
Enterocytozoon bieneusi
. SSF wetland had the highest pathogen removal rate (i.e.,
Cryptosporidium
, 97.4%;
G. duodenalis
, 95.4%); however, most of these values for FSF were in the negative area (mean, −84.0%), meaning that more pathogens were discharged by FSF wetlands than were delivered to wetlands with incoming wastewater. We demonstrate here that (i) the composition of human enteropathogens in wastewater entering and leaving SSF and FSF wetlands is highly complex and dynamic, (ii) the removal and inactivation of human-pathogenic microorganisms were significantly higher at the SSF wetland, (iii) FSF wetlands may not always provide sufficient remediation for human enteropathogens, (iv) wildlife can contribute a substantial load of human zoonotic pathogens to wetlands, (v) most of the pathogens discharged by wetlands were viable, (vi) large volumes of wetland effluents can contribute to contamination of surface waters used for recreation and drinking water abstraction and therefore represent a serious public health threat, and (vii) even with the best pathogen removal rates achieved by SSF wetland, the reduction of pathogens was not enough for a safety reuse of the reclaimed water. To our knowledge, this is the first report of
C. meleagridis
from Ireland.
Mn(II) Oxidation Is Catalyzed by Heme Peroxidases in “
Aurantimonas manganoxydans
” Strain SI85-9A1 and
Erythrobacter
sp. Strain SD-21
Authors: Anderson C. R. , Johnson H. A. , Caputo N. , Davis R. E. , Torpey J. W. , Tebo B. M.
Published: May 2009

A new type of manganese-oxidizing enzyme has been identified in two alphaproteobacteria, “
Aurantimonas manganoxydans
” strain SI85-9A1 and
Erythrobacter
sp. strain SD-21. These proteins were identified by tandem mass spectrometry of manganese-oxidizing bands visualized by native polyacrylamide gel electrophoresis in-gel activity assays and fast protein liquid chromatography-purified proteins. Proteins of both alphaproteobacteria contain animal heme peroxidase and hemolysin-type calcium binding domains, with the 350-kDa active Mn-oxidizing protein of
A. manganoxydans
containing stainable heme. The addition of both Ca
2+
ions and H
2
O
2
to the enriched protein from
Aurantimonas
increased manganese oxidation activity 5.9-fold, and the highest activity recorded was 700 μM min
−1
mg
−1
. Mn(II) is oxidized to Mn(IV) via an Mn(III) intermediate, which is consistent with known manganese peroxidase activity in fungi. The Mn-oxidizing protein in
Erythrobacter
sp. strain SD-21 is 225 kDa and contains only one peroxidase domain with strong homology to the first 2,000 amino acids of the peroxidase protein from
A. manganoxydans
. The heme peroxidase has tentatively been named MopA (manganese-oxidizing peroxidase) and sheds new light on the molecular mechanism of Mn oxidation in prokaryotes.
Distinct Roles for VeA and LaeA in Development and Pathogenesis ofAspergillus flavus
Authors: Amaike Saori , Keller Nancy P.
Published: May 2009

Aspergillus flavus, a mycotoxigenic filamentous fungus, colonizes several important agricultural crops, such as maize and peanuts. Two proteins, VeA and LaeA, known to form a nuclear complex inAspergillus nidulanshave been found to positively regulate developmental processes in severalAspergillusspecies. Here, an examination of near-isogenicA. flavusmutants differing in copy number ofveAandlaeAalleles (0, 1, or at least 2 each) revealed critical roles for VeA and LaeA inA. flavusdevelopment and seed colonization. In contrast to the wild type, both null mutants were unable to metabolize host cell lipid reserves and were inhibited by oleic acid in growth assays. The copy number of LaeA but not VeA appeared critical for a density-dependent sclerotial-to-conidial shift, since the multicopylaeA(MClaeA) strain produced relatively constant sclerotial numbers with increasing population size rather than showing the decrease in sclerotia seen in both the wild-type and MCveAstrains. The MCveA-laeAstrain yielded an intermediate phenotype. This study revealed unique roles of VeA and LaeA in seed pathogenesis and fungal biology, distinct from their cooperative regulatory functions in aflatoxin and sclerotial development.
Characterization of a REG/PA28 Proteasome Activator Homolog in
Dictyostelium discoideum
Indicates that the Ubiquitin- and ATP-Independent REGγ Proteasome Is an Ancient Nuclear Protease
Authors: Masson Patrick , Lundin Daniel , Söderbom Fredrik , Young Patrick
Published: May 2009

The nuclear proteasome activator REGγ/PA28γ is an ATP- and ubiquitin-independent activator of the 20S proteasome and has been proposed to degrade and thereby regulate both a key human oncogene, encoding the coactivator SRC-3/AIB1, and the cyclin-dependent kinase inhibitor p21 (Waf/Cip1). We report the identification and characterization of a PA28/REG homolog in
Dictyostelium
. Association of a recombinant
Dictyostelium
REG with the purified
Dictyostelium
20S proteasome led to the preferential stimulation of the trypsin-like proteasome peptidase activity. Immunolocalization studies demonstrated that the proteasome activator is localized to the nucleus and is present in growing as well as starving
Dictyostelium
cells. Our results indicate that the
Dictyostelium
PA28/REG activator can stimulate both the trypsin-like and chymotrypsin-like activities of the 20S proteasome and supports the idea that the REGγ-20S proteasome represents an early unique nuclear degradation pathway for eukaryotic cells.
Gα and Gβ Proteins Regulate the Cyclic AMP Pathway That Is Required for Development and Pathogenicity of the Phytopathogen
Mycosphaerella graminicola
Authors: Mehrabi Rahim , M'Barek Sarrah Ben , van der Lee Theo A. J. , Waalwijk Cees , de Wit Pierre J. G. M. , Kema Gerrit H. J.
Published: May 2009

We identified and functionally characterized genes encoding three Gα proteins and one Gβ protein in the dimorphic fungal wheat pathogen
Mycosphaerella graminicola
, which we designated
MgGpa1
,
MgGpa2
,
MgGpa3
, and
MgGpb1
, respectively. Sequence comparisons and phylogenetic analyses showed that MgGPA1 and MgGPA3 are most related to the mammalian Gα
i
and Gα
s
families, respectively, whereas MgGPA2 is not related to either of these families. On potato dextrose agar (PDA) and in yeast glucose broth (YGB),
MgGpa1
mutants produced significantly longer spores than those of the wild type (WT), and these developed into unique fluffy mycelia in the latter medium, indicating that this gene negatively controls filamentation.
MgGpa3
mutants showed more pronounced yeast-like growth accompanied with hampered filamentation and secreted a dark-brown pigment into YGB. Germ tubes emerging from spores of
MgGpb1
mutants were wavy on water agar and showed a nested type of growth on PDA that was due to hampered filamentation, numerous cell fusions, and increased anastomosis. Intracellular cyclic AMP (cAMP) levels of
MgGpb1
and
MgGpa3
mutants were decreased, indicating that both genes positively regulate the cAMP pathway, which was confirmed because the WT phenotype was restored by adding cAMP to these mutant cultures. The cAMP levels in
MgGpa1
mutants and the WT were not significantly different, suggesting that this gene might be dispensable for cAMP regulation. In planta assays showed that mutants of
MgGpa1
,
MgGpa3
, and
MgGpb1
are strongly reduced in pathogenicity. We concluded that the heterotrimeric G proteins encoded by
MgGpa3
and
MgGpb1
regulate the cAMP pathway that is required for development and pathogenicity in
M. graminicola
.
Increased [
PSI

+

] Appearance by Fusion of Rnq1 with the Prion Domain of Sup35 in
Saccharomyces cerevisiae
Authors: Choe Young-Jun , Ryu Yangkyun , Kim Hyun-Jin , Seok Yeong-Jae
Published: May 2009

During propagation, yeast prions show a strict sequence preference that confers the specificity of prion assembly. Although propagations of [
PSI
+
] and [
RNQ
+
] are independent of each other, the appearance of [
PSI
+
] is facilitated by the presence of [
RNQ
+
]. To explain the [
RNQ
+
] effect on the appearance of [
PSI
+
], the cross-seeding model was suggested, in which Rnq1 aggregates act as imperfect templates for Sup35 aggregation. If cross-seeding events take place in the cytoplasm of yeast cells, the collision frequency between Rnq1 aggregates and Sup35 will affect the appearance of [
PSI
+
]. In this study, to address whether cross-seeding occurs in vivo, a new [
PSI
+
] induction method was developed that exploits a protein fusion between the prion domain of Sup35 (NM) and Rnq1. This fusion protein successfully joins preexisting Rnq1 aggregates, which should result in the localization of NM around the Rnq1 aggregates and hence in an increased collision frequency between NM and Rnq1 aggregates. The appearance of [
PSI
+
] could be induced very efficiently, even with a low expression level of the fusion protein. This study supports the occurrence of in vivo cross-seeding between Sup35 and Rnq1 and provides a new tool that can be used to dissect the mechanism of the de novo appearance of prions.
Glycerol-Mediated Repression of Glucose Metabolism and Glycerol Kinase as the Sole Route of Glycerol Catabolism in the Haloarchaeon
Haloferax volcanii
Authors: Sherwood Katherine E. , Cano David José , Maupin-Furlow Julie A.
Published: May 2009

Although glycerol is the primary carbon source available to halophilic heterotrophic communities, little is known regarding haloarchaeal glycerol metabolism. In this study, a gene encoding a glycerol kinase homolog (
glpK
; HVO_1541) was deleted from the genome of the haloarchaeon
Haloferax volcanii
by a markerless knockout strategy. The
glpK
mutant, KS4, readily grew on yeast extract-peptone complex medium and glucose minimal medium but was incapable of growth on glycerol. Glycerol kinase activity was dependent on the
glpK
gene and readily detected in cells grown on glucose and/or glycerol, with the activity level higher in medium supplemented with glycerol (with or without glucose) than in medium with glucose alone. An analysis of carbon utilization revealed that glycerol suppressed the metabolism of glucose in both the parent H26 and
glpK
mutant strains, with catabolite repression more pronounced in the glycerol kinase mutant. Transcripts specific for
glpK
and an upstream gene,
gpdA
, encoding a homolog of glycerol-3-phosphate dehydrogenase subunit A, were upregulated (8- and 74-fold, respectively) in the presence of glycerol and glucose compared to those in the presence of glucose alone. Furthermore,
glpK
was transcriptionally linked to the
gpdC
gene of the putative glycerol-3-phosphate dehydrogenase operon (
gpdABC
), based on the findings of reverse transcriptase PCR analysis. The results presented here provide genetic and biochemical evidence that glycerol metabolism proceeds through a glycerol kinase encoded by
glpK
and suggest that a glycerol-3-phosphate dehydrogenase encoded by the upstream
gpdABC
operon is also involved in this pathway. Furthermore, our findings reveal a unique example of glycerol-induced repression of glucose metabolism in
H. volcanii
.
Autotrophic Carbon Dioxide Assimilation in
Thermoproteales
Revisited
Authors: Ramos-Vera W. Hugo , Berg Ivan A. , Fuchs Georg
Published: May 2009

For
Crenarchaea
, two new autotrophic carbon fixation cycles were recently described.
Sulfolobales
use the 3-hydroxypropionate/4-hydroxybutyrate cycle, with acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the carboxylating enzyme.
Ignicoccus hospitalis
(
Desulfurococcales
) uses the dicarboxylate/4-hydroxybutyrate cycle, with pyruvate synthase and phosphoenolpyruvate carboxylase being responsible for CO
2
fixation. In the two cycles, acetyl-CoA and two inorganic carbons are transformed to succinyl-CoA by different routes, whereas the regeneration of acetyl-CoA from succinyl-CoA proceeds via the same route.
Thermoproteales
would be an exception to this unifying concept, since for
Thermoproteus neutrophilus
, the reductive citric acid cycle was proposed as a carbon fixation mechanism. Here, evidence is presented for the operation of the dicarboxylate/4-hydroxybutyrate cycle in this archaeon. All required enzyme activities were detected in large amounts. The key enzymes of the cycle were strongly upregulated under autotrophic growth conditions, indicating their involvement in autotrophic CO
2
fixation. The corresponding genes were identified in the genome.
14
C-labeled 4-hydroxybutyrate was incorporated into the central building blocks in accordance with the key position of this compound in the cycle. Moreover, the results of previous
13
C-labeling studies, which could be reconciled with a reductive citric acid cycle only when some assumptions were made, were perfectly in line with the new proposal. We conclude that the dicarboxylate/4-hydroxybutyrate cycle is operating in CO
2
fixation in the strict anaerobic
Thermoproteales
as well as in
Desulfurococcales
.
Characterization of an Alcohol Dehydrogenase from the Cyanobacterium
Synechocystis
sp. Strain PCC 6803 That Responds to Environmental Stress Conditions via the Hik34-Rre1 Two-Component System
Authors: Vidal Rebeca , López-Maury Luis , Guerrero Miguel G. , Florencio Francisco J.
Published: May 2009

The slr1192 (
adhA
) gene from
Synechocystis
sp. strain PCC 6803 encodes a member of the medium-chain alcohol dehydrogenase/reductase family. The gene product AdhA exhibits NADP-dependent alcohol dehydrogenase activity, acting on a broad variety of aromatic and aliphatic primary alcohols and aldehydes but not on secondary alcohols or ketones. It exhibits superior catalytic efficiency for aldehyde reduction compared to that for alcohol oxidation. The enzyme is a cytosolic protein present in photoautotrophically grown
Synechocystis
cells. The expression of AdhA is enhanced upon the exposure of cells to different environmental stresses, although it is not essential for survival even under such stress conditions. The induction of the expression of the
adhA
gene is dependent on the Hik34-Rre1 two-component system, as it is severely impaired in mutant strains lacking either the histidine kinase Hik34 or the response regulator Rre1. In vitro DNA-protein interaction analysis reveals that the response regulator Rre1 binds specifically to the promoter region of the
adhA
gene.
The C-Terminal Domain of AcrA Is Essential for the Assembly and Function of the Multidrug Efflux Pump AcrAB-TolC
Authors: Ge Qiang , Yamada Yoichi , Zgurskaya Helen
Published: May 2009

Periplasmic membrane fusion proteins (MFPs) are essential components of multidrug efflux pumps and type I protein secretion systems of gram-negative bacteria. Located in the periplasm, MFPs function by creating a physical link between inner membrane transporters and outer membrane channels. The most conserved sequence of MFPs is located in their distal C-terminal domain. However, neither the structure nor the function of this domain is known. In this study, we investigated the structural and functional role of the C-terminal domain ofEscherichia coliAcrA, a periplasmic component of the multidrug efflux pump AcrAB-TolC. Using trypsin proteolysis, we identified the proteolytically labile sites in the C-terminal domain (amino acid residues 315 to 397) of AcrA in vitro. We next used these sites as a map to evaluate the structural integrity of this domain of AcrA inside the periplasm. We found that the C-terminal domain of AcrA is protected from trypsin when the tripartite efflux pump AcrAB-TolC is assembled. In contrast, this domain remains proteolytically labile in cells producing only one of the AcrB or TolC components of the complex. Site-directed mutagenesis of 12 highly conserved amino acid residues of the C-terminal domain of AcrA showed that a single G363C substitution dramatically impairs the multidrug efflux activity of AcrAB-TolC. The G363C mutant interacts with both AcrB and TolC but fails to properly assemble into a functional complex. We conclude that the C-terminal domain of AcrA plays an important role in the assembly and function of AcrAB-TolC efflux pump.
Regulation of Gene Expression by PrrA in
Rhodobacter sphaeroides
2.4.1: Role of Polyamines and DNA Topology
Authors: Eraso Jesus M. , Kaplan Samuel
Published: May 2009

In the present study, we show in vitro binding of PrrA, a global regulator in
Rhodobacter sphaeroides
2.4.1, to the PrrA site 2, within the RSP3361 locus. Specific binding, as shown by competition experiments, requires the phosphorylation of PrrA. The binding affinity of PrrA for site 2 was found to increase 4- to 10-fold when spermidine was added to the binding reaction. The presence of extracellular concentrations of spermidine in growing cultures of
R. sphaeroides
gave rise to a twofold increase in the expression of the photosynthesis genes
pucB
and
pufB
, as well as the RSP3361 gene, under aerobic growth conditions, as shown by the use of
lacZ
transcriptional fusions, and led to the production of light-harvesting spectral complexes. In addition, we show that negative supercoiling positively regulates the expression of the RSP3361 gene, as well as
pucB
. We show the importance of supercoiling through an evaluation of the regulation of gene expression in situ by supercoiling, in the case of the former gene, as well as using the DNA gyrase inhibitor novobiocin. We propose that polyamines and DNA supercoiling act synergistically to regulate expression of the RSP3361 gene, partly by affecting the affinity of PrrA binding to the PrrA site 2 within the RSP3361 gene.
Half-Site DNA Sequence and Spacing Length Contributions to PrrA Binding to PrrA Site 2 of RSP3361 in
Rhodobacter sphaeroides
2.4.1
Authors: Eraso Jesus M. , Kaplan Samuel
Published: May 2009

The consensus DNA binding sequence for PrrA, a global regulator in
Rhodobacter sphaeroides
2.4.1, is poorly defined. We have performed mutational analysis of PrrA site 2, of the RSP3361 gene, to which PrrA binds in vitro (J. M. Eraso and S. Kaplan, J. Bacteriol. 191:4341-4352, 2009), to further define the consensus sequence for DNA binding. Two half-sites of equal length, containing 6 nucleotides each, were required for PrrA binding to this DNA sequence. Systematic nucleotide substitutions in both inverted half-sites led to a decrease in binding affinity of phosphorylated PrrA in vitro, the level of which was dependent on the substitution. The reduced binding affinities were confirmed by competition experiments and led to proportional decreases in the expression of
lacZ
transcriptional fusions to the RSP3361 gene in vivo. The 5-nucleotide spacer region between the half-sites was found to be optimal for PrrA binding to the wild-type half-sites, as shown by decreased PrrA DNA binding affinities to synthetic DNA sequences without spacer regions or with spacer regions ranging from 1 to 10 nucleotides. The synthetic spacer region alleles also showed decreased gene expression in vivo when analyzed using
lacZ
transcriptional fusions. We have studied three additional DNA sequences to which PrrA binds in vitro. They are located in the regulatory regions of genes positively regulated by PrrA and contain spacer regions with 5 or 8 nucleotides. We demonstrate that PrrA can bind in vitro to DNA sequences with different lengths in the spacer regions between the half-sites.
An Overlap between the Control of Programmed Cell Death in
Bacillus anthracis
and Sporulation
Authors: Chandramohan Lakshmi , Ahn Jong-Sam , Weaver Keith E. , Bayles Kenneth W.
Published: May 2009

The
Staphylococcus aureus cid
and
lrg
operons have been shown to control cell death and lysis in a manner thought to be analogous to programmed cell death (apoptosis) in eukaryotic organisms. Although orthologous operons are present in a wide variety of bacterial species, members of the
Bacillus cereus
group are unique in that they have a total of four
cid
-
/lrg
-like operons. Two of these operons are similar to the
S. aureus cid
and
lrg
operons, while the other two (designated
clhAB
1
and
clhAB
2
) are unique to this group. In the present study, the functions and regulation of these loci were examined. Interestingly, the
Bacillus anthracis lrgAB
mutant displayed decreased stationary-phase survival, whereas the
clhAB
2
mutant exhibited increased stationary-phase survival compared to the parental and complementation strains. However, neither mutation had a dramatic effect on murein hydrolase activity or autolysis. Furthermore, a quantitative analysis of the sporulation efficiency revealed that both mutants formed fewer spores than did the parental strain. Similar to
S. aureus
,
B. anthracis lrgAB
transcription was shown to be induced by gramicidin and CCCP, agents known to dissipate the proton motive force, in a
lytSR
-dependent manner. Northern blot analyses also demonstrated a positive role for
lytSR
in the
clhAB
2
transcription. Taken together, the results of the present study demonstrate that
B. anthracis lrgAB
and
clhAB
2
play important roles in the control of cell death and lysis and reveal a previously unrecognized role of this system in sporulation.
The Iron-Hydrogenase of
Thermotoga maritima
Utilizes Ferredoxin and NADH Synergistically: a New Perspective on Anaerobic Hydrogen Production
Authors: Schut Gerrit J. , Adams Michael W. W.
Published: May 2009

The hyperthermophilic and anaerobic bacterium
Thermotoga maritima
ferments a wide variety of carbohydrates, producing acetate, CO
2
, and H
2
. Glucose is degraded through a classical Embden-Meyerhof pathway, and both NADH and reduced ferredoxin are generated. The oxidation of these electron carriers must be coupled to H
2
production, but the mechanism by which this occurs is unknown. The trimeric [FeFe]-type hydrogenase that was previously purified from
T. maritima
does not use either reduced ferredoxin or NADH as a sole electron donor. This problem has now been resolved by the demonstration that this hydrogenase requires the presence of both electron carriers for catalysis of H
2
production. The enzyme oxidizes NADH and ferredoxin simultaneously in an approximately 1:1 ratio and in a synergistic fashion to produce H
2
. It is proposed that the enzyme represents a new class of bifurcating [FeFe] hydrogenase in which the exergonic oxidation of ferredoxin (midpoint potential, −453 mV) is used to drive the unfavorable oxidation of NADH (
E
0
′ = −320 mV) to produce H
2
(
E
0
′ = −420 mV). From genome sequence analysis, it is now clear that there are two major types of [FeFe] hydrogenases: the trimeric bifurcating enzyme and the more well-studied monomeric ferredoxin-dependent [FeFe] hydrogenase. Almost one-third of the known H
2
-producing anaerobes appear to contain homologs of the trimeric bifurcating enzyme, although many of them also harbor one or more homologs of the simpler ferredoxin-dependent hydrogenase. The discovery of the bifurcating hydrogenase gives a new perspective on our understanding of the bioenergetics and mechanism of H
2
production and of anaerobic metabolism in general.
FIBER BRAGG GRATING BASED SYSTEM FOR TEMPERATURE MEASUREMENTS
Authors: TAHIR BASHIR AHMED , ALI JALIL , ABDUL RAHMAN ROSLY
Published: May 2009

In this study, a fiber Bragg grating sensor for temperature measurement is proposed and experimentally demonstrated. In particular, we point out that the method is well-suited for monitoring temperature because they are able to withstand a high temperature environment, where standard thermocouple methods fail. The interrogation technologies of the sensor systems are all simple, low cost and effective as well. In the sensor system, fiber grating was dipped into a water beaker that was placed on a hotplate to control the temperature of water. The temperature was raised in equal increments. The sensing principle is based on tracking of Bragg wavelength shifts caused by the temperature change. So the temperature is measured based on the wavelength-shifts of the FBG induced by the heating water. The fiber grating is high temperature stable excimer-laser-induced grating and has a linear function of wavelength-temperature in the range of 0–285°C. A dynamic range of 0–285°C and a sensitivity of 0.0131 nm/°C almost equal to that of general FBG have been obtained by this sensor system. Furthermore, the correlation of theoretical analysis and experimental results show the capability and feasibility of the purposed technique.
Novel Electrolyte for Lithium Ion Batteries: Lithum Tetrafluorooxalatophosphate (LiPF4C2O4)
Authors: Xu Mengqing , Xiao Ang , Yang Li , Lucht Brett
Published: May 2009

A novel salt for lithium ion battery electrolytes, lithium tetrafluorooxalatophosphate (LiPF4(C2O4)), has been prepared and investigated. The conductivity, electrochemical stability, and cycling performance of 1.0 M LiPF4(C2O4) in 1:1:1 ethylene carbonate:dimethyl carbonate:diethyl carbonate are comparable to the industry standard LiPF6 electrolyte. However, the LiPF4(C2O4) electrolyte has much better thermal stability. LiPF4(C2O4) electrolytes are a promising alternative for lithium ion batteries.
Physical and Electrolytic Properties of Partially Fluorinated Organic Solvents and Its Application to Secondary Lithium Batteries: Partially Fluorinated Dialkoxyethanes
Authors: Sasaki Yukio , Shimazaki Gengo , Nanbu Noritoshi , Takehara Masahiro , Ue Makoto
Published: May 2009

This paper investigated the physical and electrolytic properties, and charge-discharge characteristics for secondary lithium batteries of partially fluorinated 1-ethoxy-2-methoxyethane (EME) and 1,2-diethoxyethane (DEE) derivatives newly synthesized by us. Dielectric constants of fluorinated EME and DEE derivatives are almost independent of the number of fluorine atom. Viscosities of the fluorinated derivatives with three fluorine atoms are not affected by the molecular weights of them. Donor numbers of the fluorinated derivatives were estimated by 29Si NMR chemical shifts of triphenylsilanol. Conductivities of the fluorinated derivatives are dependent on the donor numbers of them. Oxidative decomposition voltages tend to increase with increasing the number of fluorine atom. EC-based equimolar binary derivative solutions with three fluorine atoms show higher lithium electrode cycling efficiencies. In general, EC-based fluorinated EME and DEE derivative solutions show higher discharge capacities and cycling efficiencies for Li | LiCoO2 cells.
Lithium-Ion Electrolytes Containing Flame-Retardant Additives for Increased Safety Characteristics
Authors: Smith Kiah A. , Smart Marshall C. , Prakash G. K. Surya , Ratnakumar V. B.
Published: May 2009

Recent results are presented relating to the inclusion of phosphorus flame-retarding additives added to Li-ion electrolyte solutions at 5% (v/v). Such additives are expected to reduce the flammability of these electrolytes even at low levels. Emphasis was placed on determining the effect of these additives on the performance of experimental MCMB-LiNixCo1-xO2 cells and characterizing their influence upon the lithium intercalation/de-intercalation kinetics at different temperatures. The cells were subjected to electrical characterization (charge and discharge at various temperatures and rates) and electrochemical characterization (EIS, DC micropolarization, and Tafel polarization). Furthermore, the effects that these additives have on the long-term charge-discharge cycling performance are also examined.
Mixtures of Ionic Liquid in Combination with Graphite Electrodes: The Role of Electrolyte Additives and Li-salt
Authors: Lux Simon F. , Schmuck Martin , Rupp Barbara , Kern Wolfgang , Appetecchi Giovanni B. , Passerini Stefano , Winter Martin , Balducci Andrea
Published: May 2009

The role of two different Li salt, lithium hexafluorophosphate, LiPF6, and lithium bis(trifluoromethansulfonyl) imide, LiTFSI, in mixtures of two ionic liquids, N-methyl-N-butyl pyrrolidinium bis(trifluoromethansulfonyl)imide (PYR14TFSI) and N-methyl-N-propylpyrrolidinium bis(fluorosulfonyl)imide (PYR13FSI) has been investigated. The influences of the considered mixtures on the specific capacity and the cycling stability of the electrodes have been investigated at RT. The results of this investigation are presented in this manuscript.
Li/LiCoO2 Cell Performance Using Ionic Liquids Composed of N,N-Diethyl-N-methyl-N-(2-methoxyethyl)ammonium - Effect of Anionic Structure
Authors: Matsumoto Hajime , Sakaebe Hikari , Tatsumi Kuniaki
Published: May 2009

TTo elucidate the reason why a rate performance of a Li/LiCoO2 cell with the use of FSA ([(FSO2)2N]−)-ILs gave best results compare with the other ILs, we investigated anionic structure dependence of thermal properties, viscosity, conductivity, electrochemical windows. Furthermore, an effect of Li salt addition on viscosity a Li+ and an apparent charge transfer resistance (RCT) of a Li anode and a LiCoO2 composite cathode in ILs were also investigated. For this purpose, we selected N122.1O2+ (N,N‐diethyl‐N‐methyl‐N‐(2‐methoxyethyl)ammonium) as a counter cation since the cation forms ILs with various anions such as BF4−, CF3BF3−,C2F5BF3−, C0C0− (FSA−), C1C1− (TFSA−), TSAC−, C2C2− (BETA−), PF6−, and FAP−. As a results, the good performance observed in FSA-ILs was attributed to the no significant increase of the viscosity with the addition of a Li salt and the lowest RCT on the LiCoO2.
Electrochemical Lithium Insertion/Extraction for Carbon Electrodes in FSI-based Ionic Liquids
Authors: Ishikawa Masashi , Sugimoto Toshinori , Atsumi Yosuke , Yamagata Masaki , Kikuta Manabu , Ishiko Eriko , Kono Michiyuki
Published: May 2009

Introduction of bis(fluorosulfonyl)imide (FSI) as an anion to an ambient-temperature ionic liquid electrolyte based on 1-ethyl-3-methylimidazolium and lithium (Li) as cations can provide a reversible Li intercalation into a graphitized electrode, while the intercalation is completely irreversible without FSI. Laser desorption/ionization mass spectroscopy and ac impedance spectroscopy suggest that FSI-coordinated Li+ is facilely mobile even without any solvents and is reversibly accessible to a graphite interior.
Properties of BMIBF4-LiBF4 Electrolytes for Lithium Ion Batteries
Authors: Giroud Nelly , Rouault Helene , Chainet Eric , Poignet Jean-Claude
Published: May 2009

Ionic liquids (IL) present unique properties that make them interesting for one part as battery electrolyte. As a result, ionic conductivity, dynamic viscosity and electrochemical and thermal stability of 1-butyl-3-methylimidazolium tetrafluoroborate (BMIBF4) with or without lithium tetrafluoroborate (LiBF4) are studied. Thermogravimetric and differential scanning calorimetry investigations allows the observation of the degradation and glass transition temperatures. Temperature and concentration in LiBF4 dependences of ionic conductivity and dynamic viscosity are analyzed thanks to impedance spectroscopy and rheometric methods, respectively. Furthermore, fitting laws are proposed to describe the thermal behaviors. Cyclic voltammetry experiments indicate that the reduction of BMI+ on an iron working electrode occurs at 0.9 V vs. a Li+/Li reference electrode, whereas BF4- is oxidized on a platinum working electrode at 5.7 V vs. a Li+/Li. Then LiFePO4/Li4Ti5O12 batteries using BMIBF4-LiBF4 with various concentrations as the electrolyte are assembled for cycling tests.