bluuebunny/crossref_metadata_2025_split huggingface dataset:
Function of the
cypX
and
moxY
Genes in Aflatoxin Biosynthesis in
Aspergillus parasiticus
Authors: Wen Ying , Hatabayashi Hidemi , Arai Hatsue , Kitamoto Hiroko K. , Yabe Kimiko 
Published: June 2005

ABSTRACT

The pathway oxoaverantin (OAVN) → averufin (AVR) → hydroxyversicolorone (HVN) → versiconal hemiacetal acetate (VHA) is involved in aflatoxin biosynthesis, and the
cypX
and
moxY
genes, which are present in the aflatoxin gene cluster, have been previously suggested to be involved in this pathway. To clarify the function of these two genes in more detail, we disrupted the genes in aflatoxigenic
Aspergillus parasiticus
NRRL 2999. The
cypX
-deleted mutant lost aflatoxin productivity and accumulated AVR in the mycelia. Although this mutant converted HVN, versicolorone (VONE), VHA, and versiconol acetate (VOAc) to aflatoxins in feeding experiments, it could not produce aflatoxins from either OAVN or AVR. The
moxY
-deleted mutant also lost aflatoxin productivity, whereas it newly accumulated HVN and VONE. In feeding experiments, this mutant converted either VHA or VOAc to aflatoxins but did not convert OAVN, AVR, HVN, or VONE to aflatoxins. These results demonstrated that
cypX
encodes AVR monooxygenase, catalyzing the reaction from AVR to HVN, and
moxY
encodes HVN monooxygenase, catalyzing a Baeyer-Villiger reaction from HVN to VHA as well as from VONE to VOAc. In this work, we devised a simple and rapid method to extract DNA from many fungi for PCR analyses in which cell disruption with a shaker and phenol extraction were combined.
The HtrA Protease of
Campylobacter jejuni
Is Required for Heat and Oxygen Tolerance and for Optimal Interaction with Human Epithelial Cells
Authors: Brøndsted Lone , Andersen Marianne Thorup , Parker Mary , Jørgensen Kirsten , Ingmer Hanne 
Published: June 2005

ABSTRACT

Campylobacter jejuni
is a predominant cause of food-borne bacterial gastroenteritis in the developed world. We have investigated the importance of a homologue of the periplasmic HtrA protease in
C. jejuni
stress tolerance. A
C. jejuni htrA
mutant was constructed and compared to the parental strain, and we found that growth of the mutant was severely impaired both at 44°C and in the presence of the tRNA analogue puromycin. Under both conditions, the level of misfolded protein is known to increase, and we propose that the heat-sensitive phenotype of the
htrA
mutant is caused by an accumulation of misfolded protein in the periplasm. Interestingly, we observed that the level of the molecular chaperones DnaK and ClpB was increased in the
htrA
mutant, suggesting that accumulation of nonnative proteins in the periplasm induces the expression of cytoplasmic chaperones. While lack of HtrA reduces the oxygen tolerance of
C. jejuni
, the
htrA
mutant was not sensitive to compounds that increase the formation of oxygen radicals, such as paraquat, cumene hydroperoxide, and H
2
O
2
. Using tissue cultures of human epithelial cells (INT407), we found that the
htrA
mutant adhered to and invaded human epithelial cells with a decreased frequency compared to the wild-type strain. This defect may be a consequence of the observed altered morphology of the
htrA
mutant. Thus, our results suggest that in
C. jejuni
, HtrA is important for growth during stressful conditions and has an impact on virulence.
Effects of Deregulation of Methionine Biosynthesis on Methionine Excretion in
Escherichia coli
Authors: Usuda Yoshihiro , Kurahashi Osamu 
Published: June 2005

ABSTRACT

Several regulators of methionine biosynthesis have been reported in
Escherichia coli
, which might represent barriers to the production of excess
l
-methionine (Met). In order to examine the effects of these factors on Met biosynthesis and metabolism, deletion mutations of the methionine repressor (
metJ
) and threonine biosynthetic (
thrBC
) genes were introduced into the W3110 wild-type strain of
E. coli
. Mutations of the
metK
gene encoding
S
-adenosylmethionine synthetase, which is involved in Met metabolism, were detected in 12 norleucine-resistant mutants. Three of the mutations in the
metK
structural gene were then introduced into
metJ
and
thrBC
double-mutant strains; one of the resultant strains was found to accumulate 0.13 g/liter Met. Mutations of the
metA
gene encoding homoserine succinyltransferase were detected in α-methylmethionine-resistant mutants, and these mutations were found to encode feedback-resistant enzymes in a
14
C-labeled homoserine assay. Three
metA
mutations were introduced, using expression plasmids, into an
E. coli
strain that was shown to accumulate 0.24 g/liter Met. Combining mutations that affect the deregulation of Met biosynthesis and metabolism is therefore an effective approach for the production of Met-excreting strains.
Inhibition of Efflux Transporter-Mediated Fungicide Resistance in
Pyrenophora tritici-repentis
by a Derivative of 4′-Hydroxyflavone and Enhancement of Fungicide Activity
Authors: Reimann Sven , Deising Holger B. 
Published: June 2005

ABSTRACT

Populations of the causal agent of wheat tan spot,
Pyrenophora tritici-repentis
, that are collected from fields frequently treated with reduced fungicide concentrations have reduced sensitivity to strobilurin fungicides and azole fungicides (C
14
-demethylase inhibitors). Energy-dependent efflux transporter activity can be induced under field conditions and after in vitro application of sublethal amounts of fungicides. Efflux transporters can mediate cross-resistance to a number of fungicides that belong to different chemical classes and have different modes of action. Resistant isolates can grow on substrata amended with fungicides and can infect plants treated with fungicides at levels above recommended field concentrations. We identified the hydroxyflavone derivative 2-(4-ethoxy-phenyl)-chromen-4-one as a potent inhibitor of energy-dependent fungicide efflux transporters in
P. tritici-repentis
. Application of this compound in combination with fungicides shifted fungicide-resistant
P. tritici-repentis
isolates back to normal sensitivity levels and prevented infection of wheat leaves. These results highlight the role of energy-dependent efflux transporters in fungicide resistance and could enable a novel disease management strategy based on the inhibition of fungicide efflux to be developed.
Physiological Characterization of the
ARO10
-Dependent, Broad-Substrate-Specificity 2-Oxo Acid Decarboxylase Activity of
Saccharomyces cerevisiae
Authors: Vuralhan Zeynep , Luttik Marijke A. H. , Tai Siew Leng , Boer Viktor M. , Morais Marcos A. , Schipper Dick , Almering Marinka J. H. , Kötter Peter , Dickinson J. Richard , Daran Jean-Marc , Pronk Jack T. 
Published: June 2005

ABSTRACT

Aerobic, glucose-limited chemostat cultures of
Saccharomyces cerevisiae
CEN.PK113-7D were grown with different nitrogen sources. Cultures grown with phenylalanine, leucine, or methionine as a nitrogen source contained high levels of the corresponding fusel alcohols and organic acids, indicating activity of the Ehrlich pathway. Also, fusel alcohols derived from the other two amino acids were detected in the supernatant, suggesting the involvement of a common enzyme activity. Transcript level analysis revealed that among the five thiamine-pyrophospate-dependent decarboxylases (
PDC1
,
PDC5
,
PDC6
,
ARO10
, and
THI3
), only
ARO10
was transcriptionally up-regulated when phenylalanine, leucine, or methionine was used as a nitrogen source compared to growth on ammonia, proline, and asparagine. Moreover, 2-oxo acid decarboxylase activity measured in cell extract from CEN.PK113-7D grown with phenylalanine, methionine, or leucine displayed similar broad-substrate 2-oxo acid decarboxylase activity. Constitutive expression of
ARO10
in ethanol-limited chemostat cultures in a strain lacking the five thiamine-pyrophosphate-dependent decarboxylases, grown with ammonia as a nitrogen source, led to a measurable decarboxylase activity with phenylalanine-, leucine-, and methionine-derived 2-oxo acids. Moreover, even with ammonia as the nitrogen source, these cultures produced significant amounts of the corresponding fusel alcohols. Nonetheless, the constitutive expression of
ARO10
in an isogenic wild-type strain grown in a glucose-limited chemostat with ammonia did not lead to any 2-oxo acid decarboxylase activity. Furthermore, even when
ARO10
was constitutively expressed, growth with phenylalanine as the nitrogen source led to increased decarboxylase activities in cell extracts. The results reported here indicate the involvement of posttranscriptional regulation and/or a second protein in the
ARO10
-dependent, broad-substrate-specificity decarboxylase activity.
Cooperative Effect of Two Surface Amino Acid Mutations (Q252L and E170K) in Glucose Dehydrogenase from
Bacillus megaterium
IWG3 on Stabilization of Its Oligomeric State
Authors: Baik Sang-Ho , Michel Fabrice , Aghajari Nushin , Haser Richard , Harayama Shigeaki 
Published: June 2005

ABSTRACT

A thermostable glucose dehydrogenase (GlcDH) mutant of
Bacillus megaterium
IWG3 harboring the Q252L substitution (Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Biol. Chem.
264:
6381-6385, 1989) is stable at pH values above 9, but only in the presence of 2 M NaCl. Another GlcDH mutant exhibiting increased stability at an alkaline pH in the absence of NaCl has been isolated previously (S.-H. Baik, T. Ide, H. Yoshida, O. Kagami, and S. Harayama, Appl. Microbiol. Biotechnol.
61:
329-335, 2003). This mutant had two amino acid substitutions, Q252L and E170K. In the present study, we characterized three GlcDH mutants harboring the substitutions Q252L, E170K, and Q252L/E170K under low-salt conditions. The GlcDH mutant harboring two substitutions, Q252L/E170K, was stable, but mutants harboring a single substitution, either Q252L or E170K, were unstable at an alkaline pH. Gel filtration chromatography analyses demonstrated that the oligomeric state of the Q252/E170K enzyme was stable, while the tetramers of the enzymes harboring a single substitution (Q252L or E170K) dissociated into dimers at an alkaline pH. These results indicated that the Q252L and E170K substitutions synergistically strengthened the interaction at the dimer-dimer interface. The crystal structure of the E170K/Q252L mutant, determined at 2.0-Å resolution, showed that residues 170 and 252 are located in a hydrophobic cavity at the subunit-subunit interface. We concluded that these residues in the wild-type enzyme have thermodynamically unfavorable effects, while the Q252L and E170K substitutions increase the subunit-subunit interactions by stabilizing the hydrophobic cavity.
Evolutionary Relationships of Three New Species of
Enterobacteriaceae
Living as Symbionts of Aphids and Other Insects
Authors: Moran Nancy A. , Russell Jacob A. , Koga Ryuichi , Fukatsu Takema 
Published: June 2005

ABSTRACT

Ecological studies on three bacterial lineages symbiotic in aphids have shown that they impose a variety of effects on their hosts, including resistance to parasitoids and tolerance to heat stress. Phylogenetic analyses of partial sequences of
gyrB
and
recA
are consistent with previous analyses limited to 16S rRNA gene sequences and yield improved confidence of the evolutionary relationships of these symbionts. All three symbionts are in the
Enterobacteriaceae
. One of the symbionts, here given the provisional designation “
Candidatus
Serratia symbiotica,” is a
Serratia
species that has acquired a symbiotic lifestyle. The other two symbionts, here designated “
Candidatus
Hamiltonella defensa” and “
Candidatus
Regiella insecticola,” are sister groups to one another and together show a relationship to species of
Photorhabdus
.
Complete Genome Sequence of φHSIC, a Pseudotemperate Marine Phage of
Listonella pelagia
Authors: Paul John H. , Williamson Shannon J. , Long Amy , Authement R. Nathan , John David , Segall Anca M. , Rohwer Forest L. , Androlewicz Matthew , Patterson Stacey 
Published: June 2005

ABSTRACT

The genome for the marine pseudotemperate member of the
Siphoviridae
φHSIC has been sequenced using a combination of linker amplification library construction, restriction digest library construction, and primer walking. φHSIC enters into a pseudolysogenic relationship with its host,
Listonella pelagia
, characterized by sigmoidal growth curves producing >10
9
cells/ml and >10
11
phage/ml. The genome (37,966 bp; G+C content, 44%) contained 47 putative open reading frames (ORFs), 17 of which had significant BLASTP hits in GenBank, including a β subunit of DNA polymerase III, a helicase, a helicase-like subunit of a resolvasome complex, a terminase, a tail tape measure protein, several phage-like structural proteins, and 1 ORF that may assist in host pathogenicity (an ADP ribosyltransferase). The genome was circularly permuted, with no physical ends detected by sequencing or restriction enzyme digestion analysis, and lacked a cos site. This evidence is consistent with a headful packaging mechanism similar to that of
Salmonella
phage P22 and
Shigella
phage Sf6. Because none of the phage-like ORFs were closely related to any existing phage sequences in GenBank (i.e., none more than 62% identical and most <25% identical at the amino acid level), φHSIC is unique among phages that have been sequenced to date. These results further emphasize the need to sequence phages from the marine environment, perhaps the largest reservoir of untapped genetic information.
Involvement of Bacterial Quorum-Sensing Signals in Spoilage of Bean Sprouts
Authors: Rasch Maria , Andersen Jens Bo , Nielsen Kristian Fog , Flodgaard Lars Ravn , Christensen Henrik , Givskov Michael , Gram Lone 
Published: June 2005

ABSTRACT

Bacterial communication signals, acylated homoserine lactones (AHLs), were extracted from samples of commercial bean sprouts undergoing soft-rot spoilage. Bean sprouts produced in the laboratory did not undergo soft-rot spoilage and did not contain AHLs or AHL-producing bacteria, although the bacterial population reached levels similar to those in the commercial sprouts, 10
8
to 10
9
CFU/g. AHL-producing bacteria (
Enterobacteriaceae
and pseudomonads) were isolated from commercial sprouts, and strains that were both proteolytic and pectinolytic were capable of causing soft-rot spoilage in bean sprouts. Thin-layer chromatography and liquid chromatography-high-resolution mass spectrometry revealed the presence of
N
-3-oxo-hexanoyl-
l
-homoserine lactone in spoiled bean sprouts and in extracts from pure cultures of bacteria. During normal spoilage, the pH of the sprouts increased due to proteolytic activity, and the higher pH probably facilitated the activity of pectate lyase. The AHL synthetase gene (
I
gene) from a spoilage
Pectobacterium
was cloned, sequenced, and inactivated in the parent strain. The predicted amino acid sequence showed 97% homology to HslI and CarI in
Erwinia carotovora
. Spoilage of laboratory bean sprouts inoculated with the AHL-negative mutant was delayed compared to sprouts inoculated with the wild type, and the AHL-negative mutant did not cause the pH to rise. Compared to the wild-type strain, the AHL-negative mutant had significantly reduced protease and pectinase activities and was negative in an iron chelation (siderophore) assay. This is the first study demonstrating AHL regulation of iron chelation in
Enterobacteriaceae
. The present study clearly demonstrates that the bacterial spoilage of some food products is influenced by quorum-sensing-regulated phenotypes, and understanding these processes may be useful in the development of novel food preservation additives that specifically block the quorum-sensing systems.
Development and Evaluation of a Broadly Reactive TaqMan Assay for Rapid Detection of Hepatitis A Virus
Authors: Jothikumar N. , Cromeans T. L. , Sobsey M. D. , Robertson B. H. 
Published: June 2005

ABSTRACT
Primers and a TaqMan probe for the 5′-untranslated region (UTR) of the hepatitis A virus (HAV) genome were designed and evaluated. The assay detected 0.5 infectious units of HAV and 40 copies of a synthetic transcript and provides an important screening tool for rapid quantitative HAV detection in clinical or environmental samples.
Evidence for the Presence of an Alternative Glucose Transport System in
Clostridium beijerinckii
NCIMB 8052 and the Solvent-Hyperproducing Mutant BA101
Authors: Lee Jieun , Mitchell Wilfrid J. , Tangney Martin , Blaschek H. P. 
Published: June 2005

ABSTRACT

The effects of substrate analogs and energy inhibitors on glucose uptake and phosphorylation by
Clostridium beijerinckii
provide evidence for the operation of two uptake systems: a previously characterized phosphoenolpyruvate-dependent phosphotransferase system (PTS) and a non-PTS system probably energized by the transmembrane proton gradient. In both wild-type
C. beijerinckii
NCIMB 8052 and the butanol-hyperproducing mutant BA101, PTS activity declined at the end of exponential growth, while glucokinase activity increased in the later stages of fermentation. The non-PTS uptake system, together with enhanced glucokinase activity, may provide an explanation for the ability of the mutant to utilize glucose more effectively during fermentation despite the fact that it is partially defective in PTS activity.
Deletion of the
datA
Site Does Not Affect Once-per-Cell-Cycle Timing but Induces Rifampin-Resistant Replication
Authors: Morigen  , Molina Felipe , Skarstad Kirsten 
Published: June 2005

ABSTRACT

In
Escherichia coli
, three mechanisms have been proposed to maintain proper regulation of replication so that initiation occurs once, and only once, per cell cycle. First, newly formed origins are inactivated by sequestration; second, the initiator, DnaA, is inactivated by the Hda protein at active replication forks; and third, the level of free DnaA protein is reduced by replication of the
datA
site. The
datA
site titrates unusually large amounts of DnaA and it has been reported that reinitiation, and thus asynchrony of replication, occurs in cells lacking this site. Here, we show that reinitiation in Δ
datA
cells does not occur during exponential growth and that an apparent asynchrony phenotype results from the occurrence of rifampin-resistant initiations. This shows that the
datA
site is not required to prevent reinitiation and limit initiation of replication to once per generation. The
datA
site may, however, play a role in timing of initiation relative to cell growth. Inactivation of active ATP-DnaA by the Hda protein and the sliding clamp of the polymerase was found to be required to prevent reinitiation and asynchrony of replication.
Complete Nucleotide Sequence of the LE1 Prophage from the SpirocheteLeptospira biflexaand Characterization of Its Replication and Partition Functions
Authors: Bourhy Pascale , Frangeul Lionel , Couvé Elisabeth , Glaser Philippe , Saint Girons Isabelle , Picardeau Mathieu 
Published: June 2005

ABSTRACTThe first and, to date, only extrachromosomal circular replicon identified in the spirocheteLeptospirais the LE1 prophage fromLeptospira biflexa. The 74-kb LE1 genome has a GC content of 36%, which is similar to the GC content ofLeptospiraspp. Most of the 79 predicted open reading frames (ORFs) showed no similarities to known ORFs. However 21 ORFs appeared to be organized in clusters that could code for head and tail structural proteins and immunity repressor proteins. In addition, the pattern of gene expression showed that several LE1 genes are expressed specifically either in LE1 prophage or inL. biflexalate after infection. Since the LE1 prophage replicates autonomously as a circular replicon inL. biflexa, we were able to engineer anL. biflexa-Escherichia colishuttle vector from a 5.3-kb DNA fragment of LE1 (Saint Girons et al., J. Bacteriol.182:5700-5705, 2000), opening this genus to genetic manipulation. In this study, base compositional asymmetry confirms the location of the LE1 replication region and suggests that LE1 replicates via a bidirectional Θ-like replication mechanism from this unique origin. By subcloning experiments, the replication region can be narrowed down to a 1-kb region. This minimal replication region consists of arepencoding a protein of 180 amino acids. Upstream fromrep, putative partitioning genes, calledparAandparB, were found to be similar to theparloci inBorreliaplasmids. A significant increase of plasmid stability inL. biflexacan be seen only when bothparAandparBare present. These results enable the construction of new shuttle vectors for studying the genetics ofLeptospiraspp. This study will also contribute to a better knowledge of phages unrelated to lambdoid phages.
Temperature-Dependent Conjugative Transfer of R27: Role of Chromosome- and Plasmid-Encoded Hha and H-NS Proteins
Authors: Forns Núria , Baños Rosa C. , Balsalobre Carlos , Juárez Antonio , Madrid Cristina 
Published: June 2005

ABSTRACTIncHI plasmids encode multiple-antibiotic resistance inSalmonella entericaserovar Typhi. These plasmids have been considered to play a relevant role in the persistence and reemergence of this microorganism. The IncHI1 plasmid R27, which can be considered the prototype of IncHI plasmids, is thermosensitive for transfer. Conjugation frequency is highest at low temperature (25 to 30°C), decreasing when temperature increases. R27 codifies an H-NS-like protein (open reading frame 164 [ORF164]) and an Hha-like protein (ORF182). The H-NS and Hha proteins participate in the thermoregulation of gene expression inEscherichia coli. Here we investigated the hypothetical role of such proteins in thermoregulation of R27 conjugation. At a nonpermissive temperature (33°C), transcription of several ORFs in both transfer region 1 (Tra1) and Tra2 from R27 is upregulated in cells depleted of Hha-like and H-NS-like proteins. Both chromosome- and plasmid-encoded Hha and H-NS proteins appear to potentially modulate R27 transfer. The function of R27-encoded Hha-like and H-NS proteins is not restricted to modulation of R27 transfer. Different mutant phenotypes associated with both chromosomalhhaandhnsmutations are compensated in cells harboring R27.
Transcriptional Regulation of the Flavohemoglobin Gene for Aerobic Nitric Oxide Detoxification by the Second Nitric Oxide-Responsive Regulator of
Pseudomonas aeruginosa
Authors: Arai Hiroyuki , Hayashi Michiko , Kuroi Azusa , Ishii Masaharu , Igarashi Yasuo 
Published: June 2005

ABSTRACT

The regulatory gene for a σ
54
-dependent-type transcriptional regulator,
fhpR
, is located upstream of the
fhp
gene for flavohemoglobin in
Pseudomonas aeruginosa
. Transcription of
fhp
was induced by nitrate, nitrite, nitric oxide (NO), and NO-generating reagents. Analysis of the
fhp
promoter activity in mutant strains deficient in the denitrification enzymes indicated that the promoter was regulated by NO or related reactive nitrogen species. The NO-responsive regulation was operative in a mutant strain deficient in DNR (dissimilatory nitrate respiration regulator), which is the NO-responsive regulator required for expression of the denitrification genes. A binding motif for σ
54
was found in the promoter region of
fhp
, but an FNR (fumarate nitrate reductase regulator) box was not. The
fhp
promoter was inactive in the
fhpR
or
rpoN
mutant strain, suggesting that the NO-sensing regulation of the
fhp
promoter was mediated by FhpR. The DNR-dependent denitrification promoters (
nirS
,
norC
, and
nosR
) were active in the
fhpR
or
rpoN
mutants. These results indicated that
P. aeruginosa
has at least two independent NO-responsive regulatory systems. The
fhp
or
fhpR
mutant strains showed sensitivity to NO-generating reagents under aerobic conditions but not under anaerobic conditions. These mutants also showed significantly low aerobic NO consumption activity, indicating that the physiological role of flavohemoglobin in
P. aeruginosa
is detoxification of NO under aerobic conditions.