bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of Ichip for High-Throughput
In Situ
Cultivation of “Uncultivable” Microbial Species
Authors: Nichols D. , Cahoon N. , Trakhtenberg E. M. , Pham L. , Mehta A. , Belanger A. , Kanigan T. , Lewis K. , Epstein S. S. 
Published: February 2010

ABSTRACT

One of the oldest unresolved microbiological phenomena is why only a small fraction of the diverse microbiological population grows on artificial media. The “uncultivable” microbial majority arguably represents our planet's largest unexplored pool of biological and chemical novelty. Previously we showed that species from this pool could be grown inside diffusion chambers incubated
in situ
, likely because diffusion provides microorganisms with their naturally occurring growth factors. Here we utilize this approach and develop a novel high-throughput platform for parallel cultivation and isolation of previously uncultivated microbial species from a variety of environments. We have designed and tested an isolation chip (ichip) composed of several hundred miniature diffusion chambers, each inoculated with a single environmental cell. We show that microbial recovery in the ichip exceeds manyfold that afforded by standard cultivation, and the grown species are of significant phylogenetic novelty. The new method allows access to a large and diverse array of previously inaccessible microorganisms and is well suited for both fundamental and applied research.
Activation of the Ustilagic Acid Biosynthesis Gene Cluster in
Ustilago maydis
by the C
2
H
2
Zinc Finger Transcription Factor Rua1
Authors: Teichmann Beate , Liu Lidan , Schink Kay Oliver , Bölker Michael 
Published: February 2010

ABSTRACT

The phytopathogenic basidiomycetous fungus
Ustilago maydis
secretes, under conditions of nitrogen starvation, large amounts of the biosurfactant ustilagic acid (UA). This secreted cellobiose glycolipid is toxic for many microorganisms and confers biocontrol activity to
U. maydis
. Recently, a large gene cluster that is responsible for UA biosynthesis was identified. Here, we show that expression of all cluster genes depends on Rua1, a nuclear protein of the C
2
H
2
zinc finger family, whose gene is located within the gene cluster. While deletion of
rua1
results in complete loss of UA production, overexpression of
rua1
promotes increased UA synthesis even in the presence of a good nitrogen source. Bioinformatic analysis allowed us to identify a conserved sequence element that is present in the promoters of all structural genes involved in UA biosynthesis. Deletion analysis of several promoters within the cluster revealed that this DNA element serves as an upstream activating sequence (UAS) and mediates Rua1-dependent expression. We used the yeast one-hybrid system to demonstrate specific recognition of this DNA element by Rua1. Introduction of nucleotide exchanges into the consensus sequence interfered with Rua1-dependent activation, suggesting that this sequence element acts as a direct binding site for Rua1.
Promotion of Efficient Saccharification of Crystalline Cellulose by
Aspergillus fumigatus
Swo1
Authors: Chen Xin-ai , Ishida Nobuhiro , Todaka Nemuri , Nakamura Risa , Maruyama Jun-ichi , Takahashi Haruo , Kitamoto Katsuhiko 
Published: February 2010

ABSTRACT

Swollenin is a protein from
Trichoderma reesei
that has a unique activity for disrupting cellulosic materials, and it has sequence similarity to expansins, plant cell wall proteins that have a loosening effect that leads to cell wall enlargement. In this study we cloned a gene encoding a swollenin-like protein, Swo1, from the filamentous fungus
Aspergillus fumigatus
, and designated the gene
Afswo1
. AfSwo1 has a bimodular structure composed of a carbohydrate-binding module family 1 (CBM1) domain and a plant expansin-like domain. AfSwo1 was produced using
Aspergillus oryzae
for heterologous expression and was easily isolated by cellulose-affinity chromatography. AfSwo1 exhibited weak endoglucanase activity toward carboxymethyl cellulose (CMC) and bound not only to crystalline cellulose Avicel but also to chitin, while showing no detectable affinity to xylan. Treatment by AfSwo1 caused disruption of Avicel into smaller particles without any detectable reducing sugar. Furthermore, simultaneous incubation of AfSwo1 with a cellulase mixture facilitated saccharification of Avicel. Our results provide a novel approach for efficient bioconversion of crystalline cellulose into glucose by use of the cellulose-disrupting protein AfSwo1.
Identification of Multiresistant
Salmonella
Isolates Capable of Subsisting on Antibiotics
Authors: Barnhill Alison E. , Weeks Katherine E. , Xiong Nalee , Day Tim A. , Carlson Steve A. 
Published: February 2010

ABSTRACT

This study assessed the ability of
Salmonella
(572 isolates) to subsist on 12 different antibiotics. The majority (11/12) of the antibiotics enabled subsistence for at least 1 of 140 isolates. Furthermore, 40 isolates were able to subsist on more than one antibiotic. Antibiotic resistance and antibiotic subsistence do not appear to be equivalent.
Bacterial and Protozoal Communities and Fatty Acid Profile in the Rumen of Sheep Fed a Diet Containing Added Tannins
Authors: Vasta Valentina , Yáñez-Ruiz David R. , Mele Marcello , Serra Andrea , Luciano Giuseppe , Lanza Massimiliano , Biondi Luisa , Priolo Alessandro 
Published: February 2010

ABSTRACT

This study evaluated the effects of tannins on ruminal biohydrogenation (BH) due to shifts in the ruminal microbial environment in sheep. Thirteen lambs (45 days of age) were assigned to two dietary treatments: seven lambs were fed a barley-based concentrate (control group) while the other six lambs received the same concentrate with supplemental quebracho tannins (9.57% of dry matter). At 122 days of age, the lambs were slaughtered, and the ruminal contents were subjected to fatty acid analysis and sampled to quantify populations of
Butyrivibrio fibrisolvens
, which converts C
18:2
c
9-
c
12 (linoleic acid [LA]) to C
18:2
c
9-
t
11 (rumenic acid [RA]) and then RA to C
18:1
t
11 (vaccenic acid [VA]); we also sampled for
Butyrivibrio proteoclasticus
, which converts VA to C
18:0
(stearic acid [SA]). Tannins increased (
P
< 0.005) VA in the rumen compared to the tannin-free diet. The concentration of SA was not affected by tannins. The SA/VA ratio was lower (
P
< 0.005) for the tannin-fed lambs than for the controls, suggesting that the last step of the BH process was inhibited by tannins. The
B. proteoclasticus
population was lower (−30.6%;
P
< 0.1), and
B. fibrisolvens
and protozoan populations were higher (+107% and +56.1%, respectively;
P
< 0.05) in the rumen of lambs fed the tannin-supplemented diet than in controls. These results suggest that quebracho tannins altered BH by changing ruminal microbial populations.
Role of GlnR in Acid-Mediated Repression of Genes Encoding Proteins Involved in Glutamine and Glutamate Metabolism inStreptococcus mutans
Authors: Chen Pei-Min , Chen Yi-Ywan M. , Yu Sung-Liang , Sher Singh , Lai Chern-Hsiung , Chia Jean-San 
Published: February 2010

ABSTRACTThe acid tolerance response (ATR) is one of the major virulence traits ofStreptococcus mutans. In this study, the role of GlnR in acid-mediated gene repression that affects the adaptive ATR inS. mutanswas investigated. Using a whole-genome microarray andin silicoanalyses, we demonstrated that GlnR and the GlnR box (ATGTNAN7TNACAT) were involved in the transcriptional repression of clusters of genes encoding proteins involved in glutamine and glutamate metabolism under acidic challenge. Reverse transcription-PCR (RT-PCR) analysis revealed that the coordinated regulation of the GlnR regulon occurred 5 min after acid treatment and that prolonged acid exposure (30 min) resulted in further reduction in expression. A lower level but consistent reduction in response to acidic pH was also observed in chemostat-grown cells, confirming the negative regulation of GlnR. The repression by GlnR through the GlnR box in response to acidic pH was further confirmed in thecitBZCoperon, containing genes encoding the first three enzymes in the glutamine/glutamate biosynthesis pathway. The survival rate of the GlnR-deficient mutant at pH 2.8 was more than 10-fold lower than that in the wild-type strain 45 min after acid treatment, suggesting that the GlnR regulon participates inS. mutansATR. It is hypothesized that downregulation of the synthesis of the amino acid precursors in response to acid challenge would promote citrate metabolism to pyruvate, with the consumption of H+and potential ATP synthesis. Such regulation will ensure an optimal acid adaption inS. mutans.
Increased Crystalline Cellulose Activity via Combinations of Amino Acid Changes in the Family 9 Catalytic Domain and Family 3c Cellulose Binding Module of
Thermobifida fusca
Cel9A
Authors: Li Yongchao , Irwin Diana C. , Wilson David B. 
Published: February 2010

ABSTRACT

Amino acid modifications of the
Thermobifida fusca
Cel9A-68 catalytic domain or carbohydrate binding module 3c (CBM3c) were combined to create enzymes with changed amino acids in both domains. Bacterial crystalline cellulose (BC) and swollen cellulose (SWC) assays of the expressed and purified enzymes showed that three combinations resulted in 150% and 200% increased activity, respectively, and also increased synergistic activity with other cellulases. Several other combinations resulted in drastically lowered activity, giving insight into the need for a balance between the binding in the catalytic cleft on either side of the cleavage site, as well as coordination between binding affinity for the catalytic domain and CBM3c. The same combinations of amino acid variants in the whole enzyme, Cel9A-90, did not increase BC or SWC activity but did have higher filter paper (FP) activity at 12% digestion.
AiiM, a Novel Class of
N
-Acylhomoserine Lactonase from the Leaf-Associated Bacterium
Microbacterium testaceum
Authors: Wang Wen-Zhao , Morohoshi Tomohiro , Ikenoya Masashi , Someya Nobutaka , Ikeda Tsukasa 
Published: February 2010

ABSTRACT

N
-Acylhomoserine lactones (AHLs) are used as quorum-sensing signal molecules by many Gram-negative bacteria. We have reported that
Microbacterium testaceum
StLB037, which was isolated from the leaf surface of potato, has AHL-degrading activity. In this study, we cloned the
aiiM
gene from the genomic library of StLB037, which has AHL-degrading activity and shows high homology with the α/β hydrolase fold family from
Actinobacteria
. Purified AiiM as a maltose binding fusion protein showed high degrading activity of AHLs with both short- and long-chain AHLs with or without substitution at carbon 3. High-performance liquid chromatography analysis revealed that AiiM works as an AHL lactonase that catalyzes AHL ring opening by hydrolyzing lactones. In addition, expression of AiiM in the plant pathogen
Pectobacterium carotovorum
subsp.
carotovorum
reduced pectinase activity markedly and attenuated soft rot symptoms on potato slices. In conclusion, this study indicated that AiiM might be effective in quenching quorum sensing of
P. carotovorum
subsp.
carotovorum
.
Thiosulfate-Dependent Chemolithoautotrophic Growth of
Bradyrhizobium japonicum
Authors: Masuda Sachiko , Eda Shima , Ikeda Seishi , Mitsui Hisayuki , Minamisawa Kiwamu 
Published: February 2010

ABSTRACT

Thiosulfate-oxidizing
sox
gene homologues were found at four loci (I, II, III, and IV) on the genome of
Bradyrhizobium japonicum
USDA110, a symbiotic nitrogen-fixing bacterium in soil. In fact,
B. japonicum
USDA110 can oxidize thiosulfate and grow under a chemolithotrophic condition. The deletion mutation of the
soxY
1
gene at the
sox
locus I, homologous to the sulfur-oxidizing (Sox) system in
Alphaproteobacteria
, left
B. japonicum
unable to oxidize thiosulfate and grow under chemolithotrophic conditions, whereas the deletion mutation of the
soxY
2
gene at
sox
locus II, homologous to the Sox system in green sulfur bacteria, produced phenotypes similar to those of wild-type USDA110. Thiosulfate-dependent O
2
respiration was observed only in USDA110 and the
soxY
2
mutant and not in the
soxY
1
mutant. In the cells, 1 mol of thiosulfate was stoichiometrically converted to approximately 2 mol of sulfate and consumed approximately 2 mol of O
2
.
B. japonicum
USDA110 showed
14
CO
2
fixation under chemolithotrophic growth conditions. The CO
2
fixation of resting cells was significantly dependent on thiosulfate addition. These results show that USDA110 is able to grow chemolithoautotrophically using thiosulfate as an electron donor, oxygen as an electron acceptor, and carbon dioxide as a carbon source, which likely depends on
sox
locus I including the
soxY
1
gene on USDA110 genome. Thiosulfate oxidation capability is frequently found in members of the
Bradyrhizobiaceae
, which phylogenetic analysis showed to be associated with the presence of
sox
locus I homologues, including the
soxY
1
gene of
B. japonicum
USDA110.
Cre-
lox
-Based Method for Generation of Large Deletions within the Genomic Magnetosome Island of
Magnetospirillum gryphiswaldense
Authors: Ullrich Susanne , Schüler Dirk 
Published: February 2010

ABSTRACT

Magnetosome biomineralization and magnetotaxis in magnetotactic bacteria are controlled by numerous, mostly unknown gene functions that are predominantly encoded by several operons located within the genomic magnetosome island (MAI). Genetic analysis of magnetotactic bacteria has remained difficult and requires the development of novel tools. We established a Cre-
lox
-based deletion method which allows the excision of large genomic fragments in
Magnetospirillum gryphiswaldense
. Two conjugative suicide plasmids harboring
lox
sites that flanked the target region were subsequently inserted into the chromosome by homologous recombination, requiring only one single-crossover event, respectively, and resulting in a double cointegrate. Excision of the targeted chromosomal segment that included the inserted plasmids and their resistance markers was induced by
trans
expression of Cre recombinase, which leaves behind a scar of only a single
loxP
site. The Cre helper plasmid was then cured from the deletant strain by relief of antibiotic selection. We have used this method for the deletion of 16.3-kb, 61-kb, and 67.3-kb fragments from the genomic MAI, either in a single round or in subsequent rounds of deletion, covering a region of approximately 87 kb that comprises the
mamAB
,
mms6
, and
mamGFDC
operons. As expected, all mutants were Mag
−
and some were Mot
−
; otherwise, they showed normal growth patterns, which indicates that the deleted region is not essential for viability in the laboratory. The method will facilitate future functional analysis of magnetosome genes and also can be utilized for large-scale genome engineering in magnetotactic bacteria.
Effects of Polyphosphate Additives on
Campylobacter
Survival in Processed Chicken Exudates
Authors: Gunther Nereus W. 
Published: February 2010

ABSTRACT

Campylobacter
spp. are responsible for a large number of the bacterial food poisoning cases worldwide. Despite being sensitive to oxygen and nutritionally fastidious,
Campylobacter
spp. are able to survive in food processing environments and reach consumers in sufficient numbers to cause disease. To investigate
Campylobacter
persistence on processed chicken, exudates from chickens produced for consumer sale were collected and sterilized. Two types of exudates from chicken products were collected: enhanced, where a marinade was added to the chickens during processing, and nonenhanced, where no additives were added during processing. Exudates from enhanced chicken products examined in this study contained a mixture of polyphosphates. Exudate samples were inoculated with
Campylobacter jejuni
or
Campylobacter coli
strains and incubated under a range of environmental conditions, and viable bacteria present in the resultant cultures were enumerated. When incubated at 42°C in a microaerobic environment, exudates from enhanced chicken products resulted in increased survival of
C. jejuni
and
C. coli
compared with that in nonenhanced exudates in the range of <1 to >4 log CFU/ml. Under more relevant food storage conditions (4°C and normal atmosphere), the exudates from enhanced chicken products also demonstrated improved
Campylobacter
survival compared with that in nonenhanced exudates. Polyphosphates present in the enhanced exudates were determined to be largely responsible for the improved survival observed when the two types of exudates were compared. Therefore, polyphosphates used to enhance chicken quality aid in sustaining the numbers of
Campylobacter
bacteria, increasing the opportunity for disease via cross-contamination or improperly cooked poultry.
Rickettsia
Symbionts Cause Parthenogenetic Reproduction in the Parasitoid Wasp
Pnigalio soemius
(Hymenoptera: Eulophidae)
Authors: Giorgini M. , Bernardo U. , Monti M. M. , Nappo A. G. , Gebiola M. 
Published: February 2010

ABSTRACT

Bacteria in the genus
Rickettsia
are intracellular symbionts of disparate groups of organisms. Some
Rickettsia
strains infect vertebrate animals and plants, where they cause diseases, but most strains are vertically inherited symbionts of invertebrates. In insects
Rickettsia
symbionts are known to have diverse effects on hosts ranging from influencing host fitness to manipulating reproduction. Here we provide evidence that a
Rickettsia
symbiont causes thelytokous parthenogenesis (in which mothers produce only daughters from unfertilized eggs) in a parasitoid wasp,
Pnigalio soemius
(Hymenoptera: Eulophidae). Feeding antibiotics to thelytokous female wasps resulted in production of progeny that were almost all males. Cloning and sequencing of a fragment of the 16S rRNA gene amplified with universal primers, diagnostic PCR screening of symbiont lineages associated with manipulation of reproduction, and fluorescence
in situ
hybridization (FISH) revealed that
Rickettsia
is always associated with thelytokous
P. soemius
and that no other bacteria that manipulate reproduction are present. Molecular analyses and FISH showed that
Rickettsia
is distributed in the reproductive tissues and is transovarially transmitted from mothers to offspring. Comparison of antibiotic-treated females and untreated females showed that infection had no cost. Phylogenetic analyses of 16S rRNA and
gltA
gene sequences placed the symbiont of
P. soemius
in the bellii group and indicated that there have been two separate origins of the parthenogenesis-inducing phenotype in the genus
Rickettsia
. A possible route for evolution of induction of parthenogenesis in the two distantly related
Rickettsia
lineages is discussed.
Mapping of Vps21 and HOPS Binding Sites in Vps8 and Effect of Binding Site Mutants on Endocytic Trafficking
Authors: Pawelec Agnes , Arsić Janja , Kölling Ralf 
Published: February 2010

ABSTRACT

Vps8 is a subunit of the CORVET tethering complex, which is involved in early-to-late endosome fusion. Here, we examine the role of Vps8 in membrane fusion at late endosomes in
Saccharomyces cerevisiae
. We demonstrate that Vps8 associates with membranes and that this association is independent of the class C/HOPS core complex and, contrary to a previous report, also independent of the Rab GTPase Vps21. Our data indicate that Vps8 makes multiple contacts with membranes. One of these membrane binding regions could be mapped to the N-terminal part of the protein. By two-hybrid analysis, we obtained evidence for a physical interaction between Vps8 and the Rab5 homologue Vps21. In addition, the interaction with the HOPS core complex was confirmed by immunoprecipitation experiments. By deletion analysis, the Vps21 and HOPS binding sites were mapped in Vps8. Deletions that abrogated HOPS core complex binding had a strong effect on the turnover of the endocytic cargo protein Ste6 and on vacuolar sorting of carboxypeptidase Y. In contrast, deletions that abolished Vps21 binding showed only a modest effect. This suggests that the Vps21 interaction is not essential for endosomal trafficking but may be important for some other aspect of Vps8 function.
Use of the Kinase Inhibitor Analog 1NM-PP1 Reveals a Role for Toxoplasma gondii CDPK1 in the Invasion Step
Authors: Sugi Tatsuki , Kato Kentaro , Kobayashi Kyosuke , Watanabe Shumpei , Kurokawa Hitomi , Gong Haiyan , Pandey Kishor , Takemae Hitoshi , Akashi Hiroomi 
Published: February 2010

ABSTRACT

Toxoplasma gondii
CDPK1 (TgCDPK1) was found to be the target of the toxoplasmocidal compound 1NM-PP1. When TgCDPK1 was mutated at position 128 from glycine to methionine, resistance was gained. Inhibition of gliding motility without inhibition of micronemal secretion by 1NM-PP1 suggests a function for TgCDPK1 in gliding motility.
Metabolic and Developmental Effects Resulting from Deletion of thecitAGene Encoding Citrate Synthase in Aspergillus nidulans
Authors: Murray Sandra L. , Hynes Michael J. 
Published: February 2010

ABSTRACTCitrate synthase is a central activity in carbon metabolism. It is required for the tricarboxylic acid (TCA) cycle, respiration, and the glyoxylate cycle. InSaccharomyces cerevisiaeandArabidopsis thaliana, there are mitochondrial and peroxisomal isoforms encoded by separate genes, while inAspergillus nidulans, a single gene,citA, encodes a protein with predicted mitochondrial and peroxisomal targeting sequences (PTS). Deletion ofcitAresults in poor growth on glucose but not on derepressing carbon sources, including those requiring the glyoxylate cycle. Growth on glucose is restored by a mutation in thecreAcarbon catabolite repressor gene. Methylcitrate synthase, required for propionyl-coenzyme A (CoA) metabolism, has previously been shown to have citrate synthase activity. We have been unable to construct themcsAΔcitAΔ double mutant, and the expression ofmcsAis subject to CreA-mediated carbon repression. Therefore, McsA can substitute for the loss of CitA activity. Deletion ofcitAdoes not affect conidiation or sexual development but results in delayed conidial germination as well as a complete loss of ascospores in fruiting bodies, which can be attributed to loss of meiosis. These defects are suppressed by thecreA204mutation, indicating that McsA activity can substitute for the loss of CitA. A mutation of the putative PTS1-encoding sequence incitAhad no effect on carbon source utilization or development but did result in slower colony extension arising from single conidia or ascospores. CitA-green fluorescent protein (GFP) studies showed mitochondrial localization in conidia, ascospores, and hyphae. Peroxisomal localization was not detected. However, a very low and variable detection of punctate GFP fluorescence was sometimes observed in conidia germinated for 5 h when the mitochondrial targeting sequence was deleted.