bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biosynthesis of
Staphylococcus aureus
Autoinducing Peptides by Using the
Synechocystis
DnaB Mini-Intein
Authors: Malone Cheryl L. , Boles Blaise R. , Horswill Alexander R. 
Published: August 2007

ABSTRACT

The Agr quorum-sensing system of
Staphylococcus aureus
modulates the expression of virulence factors in response to autoinducing peptides (AIPs). The peptides are seven to nine residues in length and have the C-terminal five residues constrained in a thiolactone ring. We have developed a new method to generate AIP structures using an engineered DnaB mini-intein from
Synechocystis
sp. strain PCC6803. In the method, an oligonucleotide encoding the AIP is ligated to the intein and the fusion protein is expressed and purified by affinity chromatography. To produce the correct AIP structure, intein splicing is interrupted, allowing the cysteine side chain to catalyze thiolactone ring formation and release AIP from the resin. The technique is simple and robust, and we have successfully produced the three main classes of AIPs using the intein system. The intein-generated AIPs possessed the correct thiolactone ring modification based on biochemical analysis, and, importantly, all the samples were bioactive against
S. aureus
. The AIP activity was confirmed through Agr interference and activation profiling with developed
S. aureus
reporter strains. The simplicity of the method, benefits of DNA encoding, and scalable nature enable the production of
S. aureus
AIPs for many biological applications.
Shuffling of Promoters for Multiple Genes To Optimize Xylose Fermentation in an Engineered
Saccharomyces cerevisiae
Strain
Authors: Lu Chenfeng , Jeffries Thomas 
Published: August 2007

ABSTRACT

We describe here a useful metabolic engineering tool, multiple-gene-promoter shuffling (MGPS), to optimize expression levels for multiple genes. This method approaches an optimized gene overexpression level by fusing promoters of various strengths to genes of interest for a particular pathway. Selection of these promoters is based on the expression levels of the native genes under the same physiological conditions intended for the application. MGPS was implemented in a yeast xylose fermentation mixture by shuffling the promoters for
GND2
and
HXK2
with the genes for transaldolase (
TAL1
), transketolase (
TKL1
), and pyruvate kinase (
PYK1
) in the
Saccharomyces cerevisiae
strain FPL-YSX3. This host strain has integrated xylose-metabolizing genes, including xylose reductase, xylitol dehydrogenase, and xylulose kinase. The optimal expression levels for
TAL1
,
TKL1
, and
PYK1
were identified by analysis of volumetric ethanol production by transformed cells. We found the optimal combination for ethanol production to be
GND2
-
TAL1
-
HXK2
-
TKL1
-
HXK2
-
PYK1
. The MGPS method could easily be adapted for other eukaryotic and prokaryotic organisms to optimize expression of genes for industrial fermentation.
Multiplex PCR for Simultaneous Detection of Bacteria of the Genus
Listeria
,
Listeria monocytogenes
, and Major Serotypes and Epidemic Clones of
L. monocytogenes
Authors: Chen Yi , Knabel Stephen J. 
Published: August 2007

ABSTRACT

A multiplex PCR assay which combines detection of bacteria of the genus
Listeria
,
Listeria monocytogenes
serotypes 1/2a and 4b, and epidemic clones I, II, and III of
L. monocytogenes
was developed. The assay provides a rapid, reliable, and inexpensive method for screening and subgrouping this important food-borne pathogen.
Effect of Insect Larval Midgut Proteases on the Activity of
Bacillus thuringiensis
Cry Toxins
Authors: Fortier Mélanie , Vachon Vincent , Frutos Roger , Schwartz Jean-Louis , Laprade Raynald 
Published: August 2007

ABSTRACT

To test the possibility that proteolytic cleavage by midgut juice enzymes could enhance or inhibit the activity of
Bacillus thuringiensis
insecticidal toxins, once activated, the effects of different toxins on the membrane potential of the epithelial cells of isolated
Manduca sexta
midguts in the presence and absence of midgut juice were measured. While midgut juice had little effect on the activity of Cry1Aa, Cry1Ac, Cry1Ca, Cry1Ea, and R233A, a mutant of Cry1Aa from which one of the four salt bridges linking domains I and II of the toxin was eliminated, it greatly increased the activity of Cry1Ab. In addition, when tested in the presence of a cocktail of protease inhibitors or when boiled, midgut juice retained almost completely its capacity to enhance Cry1Ab activity, suggesting that proteases were not responsible for the stimulation. On the other hand, in the absence of midgut juice, the cocktail of protease inhibitors also enhanced the activity of Cry1Ab, suggesting that proteolytic cleavage by membrane proteases could render the toxin less effective. The lower toxicity of R233A, despite a similar in vitro pore-forming ability, compared with Cry1Aa, cannot be accounted for by an increased susceptibility to midgut proteases. Although these assays were performed under conditions approaching those found in the larval midgut, the depolarizing activities of the toxins correlated only partially with their toxicities.
Changes in Bacterial Populations and in Biphenyl Dioxygenase Gene Diversity in a Polychlorinated Biphenyl-Polluted Soil after Introduction of Willow Trees for Rhizoremediation
Authors: de Cárcer Daniel Aguirre , Martín Marta , Karlson Ulrich , Rivilla Rafael 
Published: August 2007

ABSTRACT

The aim of this study was to analyze the structural and functional changes occurring in a polychlorinated-biphenyl (PCB)-contaminated soil ecosystem after the introduction of a suitable host plant for rhizoremediation (
Salix viminalis
). We have studied the populations and phylogenetic distribution of key bacterial groups (
Alpha
- and
Betaproteobacteria
,
Acidobacteria
, and
Actinobacteria
) and the genes encoding iron-sulfur protein α (ISPα) subunits of the toluene/biphenyl dioxygenases in soil and rhizosphere by screening gene libraries using temperature gradient gel electrophoresis. The results, based on the analysis of 415 clones grouped into 133 operational taxonomic units that were sequence analyzed (>128 kbp), show that the rhizospheric bacterial community which evolved from the native soil community during the development of the root system was distinct from the soil community for all groups studied except for the
Actinobacteria. Proteobacteria
were enriched in the rhizosphere and dominated both in rhizosphere and soil. There was a higher than expected abundance of
Betaproteobacteria
in the native and in the planted PCB-polluted soil. The ISPα sequences retrieved indicate a high degree of catabolic and phylogenetic diversity. Many sequences clustered with biphenyl dioxygenase sequences from gram-negative bacteria. A distinct cluster that was composed of sequences from this study, some previously described environmental sequences, and a putative ISPα from
Sphingomonas wittichii
RW1 seems to contain greater diversity than the presently recognized toluene/biphenyl dioxygenase subfamily. Moreover, the rhizosphere selected for two ISPα sequences that accounted for almost 60% of the gene library and were very similar to sequences harbored by
Pseudomonas
species.
Identification of Indole Derivatives as Self-Growth Inhibitors of
Symbiobacterium thermophilum
, a Unique Bacterium Whose Growth Depends on Coculture with a
Bacillus
sp
Authors: Watsuji Tomo-o , Yamada Shinya , Yamabe Tomoya , Watanabe Yuka , Kato Taira , Saito Takao , Ueda Kenji , Beppu Teruhiko 
Published: August 2007

ABSTRACT

Symbiobacterium thermophilum
is a syntrophic bacterium whose growth depends on coculture with a
Bacillus
sp. Recently, we discovered that CO
2
generated by
Bacillus
is the major inducer for the growth of
S. thermophilum
; however, the evidence suggested that an additional element is required for its full growth. Here, we studied the self-growth-inhibitory substances produced by
S. thermophilum
. We succeeded in purifying two substances from an ether extract of the culture supernatant of
S. thermophilum
by multiple steps of reverse-phase chromatography. Electron ionization mass spectrometry and nuclear magnetic resonance analyses of the purified preparation identified the substances as 2,2-bis(3′-indolyl)indoxyl (BII) and 1,1-bis(3′-indolyl)ethane (BIE). The pure growth of
S. thermophilum
was inhibited by authentic BII and BIE with MICs of 12 and 7 μg/ml, respectively; however, its growth in coculture with
Bacillus
was not inhibited by BII at the saturation concentration and was inhibited by BIE with an MIC of 14 μg/ml. Both BII and BIE inhibited the growth of other microorganisms. Unexpectedly, the accumulation levels of both BII and BIE in the pure culture of
S. thermophilum
were far lower than the MICs (<0.1 μg/ml) while a marked amount of BIE (6 to 7 μg/ml) equivalent to the MIC had accumulated in the coculture. An exogenous supply of surfactin alleviated the sensitivities of several BIE-sensitive bacteria against BIE. The results suggest that
Bacillus
benefits
S. thermophilum
by detoxifying BII and BIE in the coculture. A similar mechanism may underlie mutualistic relationships between different microorganisms.
WdStuAp, an APSES Transcription Factor, Is a Regulator of Yeast-Hyphal Transitions in
Wangiella
(
Exophiala
)
dermatitidis
Authors: Wang Qin , Szaniszlo Paul J. 
Published: August 2007

ABSTRACT

APSES transcription factors are well-known regulators of fungal cellular development and differentiation. To study the function of an APSES protein in the fungus
Wangiella dermatitidis
, a conidiogenous and polymorphic agent of human phaeohyphomycosis with yeast predominance, the APSES transcription factor gene Wd
STUA
was cloned, sequenced, disrupted, and overexpressed. Analysis showed that its derived protein was most similar to the APSES proteins of other conidiogenous molds and had its APSES DNA-binding domain located in the amino-terminal half. Deletion of Wd
STUA
in
W. dermatitidis
induced convoluted instead of normal smooth colony surface growth on the rich yeast maintenance agar medium yeast extract-peptone-dextrose agar (YPDA) at 37°C. Additionally, deletion of Wd
STUA
repressed aerial hyphal growth, conidiation, and invasive hyphal growth on the nitrogen-poor, hypha-inducing agar medium potato dextrose agar (PDA) at 25°C. Ectopic overexpression of Wd
STUA
repressed the convoluted colony surface growth on YPDA at 37°C, and also strongly repressed hyphal growth on PDA at 25°C and 37°C. These new results provide additional insights into the diverse roles played by APSES factors in fungi. They also suggest that the transcription factor encoded by Wd
STUA
is both a positive and negative morphotype regulator in
W. dermatitidis
and possibly other of the numerous human pathogenic, conidiogenous fungi capable of yeast growth.
Genotypic Evolution of Azole Resistance Mechanisms in Sequential
Candida albicans
Isolates
Authors: Coste Alix , Selmecki Anna , Forche Anja , Diogo Dorothée , Bougnoux Marie-Elisabeth , d'Enfert Christophe , Berman Judith , Sanglard Dominique 
Published: August 2007

ABSTRACT

TAC1
(for transcriptional activator of
CDR
genes) is critical for the upregulation of the ABC transporters
CDR1
and
CDR2
, which mediate azole resistance in
Candida albicans
. While a wild-type
TAC1
allele drives high expression of
CDR1/2
in response to inducers, we showed previously that
TAC1
can be hyperactive by a gain-of-function (GOF) point mutation responsible for constitutive high expression of
CDR1/2
. High azole resistance levels are achieved when
C. albicans
carries hyperactive alleles only as a consequence of loss of heterozygosity (LOH) at the
TAC1
locus on chromosome 5 (Chr 5), which is linked to the mating-type-like (
MTL
) locus. Both are located on the Chr 5 left arm along with
ERG11
(target of azoles). In this work, five groups of related isolates containing azole-susceptible and -resistant strains were analyzed for the
TAC1
and
ERG11
alleles and for Chr 5 alterations. While recovered
ERG11
alleles contained known mutations, 17 new
TAC1
alleles were isolated, including 7 hyperactive alleles with five separate new GOF mutations. Single-nucleotide-polymorphism analysis of Chr 5 revealed that azole-resistant strains acquired
TAC1
hyperactive alleles and, in most cases,
ERG11
mutant alleles by LOH events not systematically including the
MTL
locus.
TAC1
LOH resulted from mitotic recombination of the left arm of Chr 5, gene conversion within the
TAC1
locus, or the loss and reduplication of the entire Chr 5. In one case, two independent
TAC1
hyperactive alleles were acquired. Comparative genome hybridization and karyotype analysis revealed the presence of isochromosome 5L [i(5L)] in two azole-resistant strains. i(5L) leads to increased copy numbers of azole resistance genes present on the left arm of Chr 5, among them
TAC1
and
ERG11
. Our work shows that azole resistance was due not only to the presence of specific mutations in azole resistance genes (at least
ERG11
and
TAC1
) but also to their increase in copy number by LOH and to the addition of extra Chr 5 copies. With the combination of these different modifications, sophisticated genotypes were obtained. The development of azole resistance in
C. albicans
is therefore a powerful instrument for generating genetic diversity.
Differential Regulation and Substrate Preferences in Two Peptide Transporters of
Saccharomyces cerevisiae
Authors: Cai Houjian , Hauser Melinda , Naider Fred , Becker Jeffrey M. 
Published: August 2007

ABSTRACT

Dal5p has been shown previously to act as an allantoate/ureidosuccinate permease and to play a role in the utilization of certain dipeptides as a nitrogen source in
Saccharomyces cerevisiae
. Here, we provide direct evidence that dipeptides are transported by Dal5p, although the affinity of Dal5p for allantoate and ureidosuccinate is higher than that for dipeptides. Allantoate, ureidosuccinate, and to a lesser extent allantoin competed with dipeptide transport by reducing the toxicity of the peptide Ala-Eth and decreasing the accumulation of [
14
C]Gly-Leu. In contrast to the well-studied di/tripeptide transporter Ptr2p, whose substrate specificity is very broad, Dal5p preferred to transport non-N-end rule dipeptides.
S. cerevisiae
W303 was sensitive to the toxic peptide Ala-Eth (non-N-end rule peptide) but not Leu-Eth (N-end rule peptide). Non-N-end rule dipeptides showed better competition with the uptake of [
14
C]Gly-Leu than N-end rule dipeptides. Similar to the regulation of
PTR2, DAL5
expression was influenced by the addition of Leu and by the
CUP9
gene. However,
DAL5
expression was downregulated in the presence of leucine and the absence of
CUP9
, whereas
PTR2
was upregulated. Toxic dipeptide and uptake assays indicated that either Ptr2p or Dal5p was predominantly used for dipeptide transport in the common laboratory strains S288c and W303, respectively. These studies highlight the complementary activities of two dipeptide transport systems under different regulatory controls in common laboratory yeast strains, suggesting that dipeptide transport pathways evolved to respond to different environmental conditions.
Host Protein Binding and Adhesive Properties of H6 and H7 Flagella of Attaching and Effacing
Escherichia coli
Authors: Erdem Ayşen L. , Avelino Fabiola , Xicohtencatl-Cortes Juan , Girón Jorge A. 
Published: August 2007

ABSTRACT

It had been suggested that the flagella of enteropathogenic
Escherichia coli
(EPEC) and enterohemorrhagic
E. coli
(EHEC) might contribute to host colonization. In this study, we set out to investigate the adhesive properties of H7 and H6 flagella. We studied the abilities of EHEC EDL933 (O157:H7) and EPEC E2348/69 (O127:H6) flagella to bind to bovine mucus, host proteins such as mucins, and extracellular matrix proteins. Through several approaches, we found that H6 and H7 flagella and their flagellin monomers bind to mucins I and II and to freshly isolated bovine mucus. A genetic approach showed that EHEC and EPEC
fliC
deletion mutants were significantly less adherent to bovine intestinal tissue than the parental wild-type strains. In addition, we found that EPEC bacteria and H6 flagella, but not EHEC, bound largely, in a dose-dependent manner, to collagen and to a lesser extent to laminin and fibronectin. We also report that EHEC O157:H7 strains agglutinate rabbit red blood cells via their flagella, a heretofore unknown phenotype in this pathogroup. Collectively, our data demonstrate that the H6 and H7 flagella possess adhesive properties, particularly the ability to bind mucins, that may contribute to colonization of mucosal surfaces.
Structure of the
Haemophilus influenzae
HMW1B Translocator Protein: Evidence for a Twin Pore
Authors: Li Huilin , Grass Susan , Wang Tao , Liu Tianbo , St. Geme Joseph W. 
Published: August 2007

ABSTRACT

Secretion of the
Haemophilus influenzae
HMW1 adhesin occurs via the two-partner secretion pathway and requires the HMW1B outer membrane translocator. HMW1B has been subjected to extensive biochemical studies to date. However, direct examination of the structure of HMW1B has been lacking, leaving fundamental questions about the oligomeric state, the membrane-embedded β-barrel domain, the approximate size of the β-barrel pore, and the mechanism of translocator activity. In the current study, examination of purified HMW1B by size exclusion chromatography and negative staining electron microscopy revealed that the predominant species was a dimer. In the presence of lipid, purified HMW1B formed two-dimensional crystalline sheets. Examination of these crystals by cryo-electron microscopy allowed determination of a projection structure of HMW1B to 10 Å resolution. The native HMW1B structure is a dimer of β-barrels, with each β-barrel measuring 40 Å by 50 Å in the two orthogonal directions and appearing largely occluded, leaving only a narrow pore. These observations suggest that HMW1B undergoes a large conformational change during translocation of the 125-kDa HMW1 adhesin.
Cwp84, a Surface-Associated Protein of
Clostridium difficile
, Is a Cysteine Protease with Degrading Activity on Extracellular Matrix Proteins
Authors: Janoir Claire , Péchiné Séverine , Grosdidier Charlotte , Collignon Anne 
Published: August 2007

ABSTRACT

Clostridium difficile
pathogenicity is mediated mainly by its A and B toxins, but the colonization process is thought to be a necessary preliminary step in the course of infection. The aim of this study was to characterize the Cwp84 protease of
C. difficile
, which is highly immunogenic in patients with
C. difficile
-associated disease and is potentially involved in the pathogenic process. Cwp84 was purified as a recombinant His-tagged protein, and specific antibodies were generated in rabbits. Treatment of multiple-band-containing eluted fractions with a reducing agent or with trypsin led to accumulation of a unique protein species with an estimated molecular mass of 61 kDa, corresponding most likely to mature autoprocessed Cwp84 (mCwp84). mCwp84 showed concentration-dependent caseinolytic activity, with maximum activity at pH 7.5. The Cwp84 activity was inhibited by various cysteine protease inhibitors, such as the specific inhibitor E64, and the anti-Cwp84-specific antibodies. Using fractionation experiments followed by immunoblot detection, the protease was found to be associated with the S-layer proteins, mostly as a nonmature species. Proteolytic assays were performed with extracellular matrix proteins to assess the putative role of Cwp84 in the pathogenicity of
C. difficile
. No degrading activity was detected with type IV collagen. In contrast, Cwp84 exhibited degrading activity with fibronectin, laminin, and vitronectin, which was neutralized by the E64 inhibitor and specific antibodies. In vivo, this proteolytic activity could contribute to the degradation of the host tissue integrity and to the dissemination of the infection.
Characterization and Biological Role of the O-Polysaccharide Gene Cluster ofYersinia enterocoliticaSerotype O:9
Authors: Skurnik Mikael , Biedzka-Sarek Marta , Lübeck Peter S. , Blom Tea , Bengoechea José Antonio , Pérez-Gutiérrez Camino , Ahrens Peter , Hoorfar Jeffrey 
Published: August 2007

ABSTRACTYersinia enterocoliticaserotype O:9 is a gram-negative enteropathogen that infects animals and humans. The role of lipopolysaccharide (LPS) inY. enterocoliticaO:9 pathogenesis, however, remains unclear. The O:9 LPS consists of lipid A to which is linked the inner core oligosaccharide, serving as an attachment site for both the outer core (OC) hexasaccharide and the O-polysaccharide (OPS; a homopolymer ofN-formylperosamine). In this work, we cloned the OPS gene cluster of O:9 and identified 12 genes organized into four operons upstream of thegndgene. Ten genes were predicted to encode glycosyltransferases, the ATP-binding cassette polysaccharide translocators, or enzymes required for the biosynthesis of GDP-N-formylperosamine. The two remaining genes within the OPS gene cluster,galFandgalU, were not ascribed a clear function in OPS biosynthesis; however, the latter gene appeared to be essential for O:9. The biological functions of O:9 OPS and OC were studied using isogenic mutants lacking one or both of these LPS parts. We showed that OPS and OC confer resistance to human complement and polymyxin B; the OPS effect on polymyxin B resistance could be observed only in the absence of OC.
Transcriptional Regulation of Stress Response and Motility Functions inHelicobacter pyloriIs Mediated by HspR and HrcA
Authors: Roncarati Davide , Danielli Alberto , Spohn Gunther , Delany Isabel , Scarlato Vincenzo 
Published: August 2007

ABSTRACTThehrcAandhspRgenes ofHelicobacter pyloriencode two transcriptional repressor proteins that negatively regulate expression of thegroES-groELandhrcA-grpE-dnaKoperons. While HspR was previously shown to bind far upstream of the promoters transcribing these operons, the binding sites of HrcA were not identified. Here, we demonstrate by footprinting analysis that HrcA binds to operator elements similar to the so-called CIRCE sequences overlapping both promoters. Binding of HspR and HrcA to their respective operators occurs in an independent manner, but the DNA binding activity of HrcA is increased in the presence of GroESL, suggesting that the GroE chaperonin system corepresses transcription together with HrcA. Comparative transcriptome analysis of the wild-type strain andhspRandhrcAsingly and doubly deficient strains revealed that a set of 14 genes is negatively regulated by the action of one or both regulators, while a set of 29 genes is positively regulated. While both positive and negative regulation of transcription by HspR and/or HrcA could be confirmed by RNA primer extension analyses for two representative genes, binding of either regulator to the promoters could not be detected, indicating that transcriptional regulation at these promoters involves indirect mechanisms. Strikingly, 14 of the 29 genes which were found to be positively regulated by HspR or HrcA code for proteins involved in flagellar biosynthesis. Accordingly, loss of motility functions was observed for HspR and HrcA single or double mutants. The possible regulatory intersections of the heat shock response and flagellar assembly are discussed.