bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transient Marker System for Iterative Gene Targeting of a Prototrophic Fungus
Authors: Nielsen Michael L. , de Jongh Willem A. , Meijer Susan L. , Nielsen Jens , Mortensen Uffe H. 
Published: October 2007

ABSTRACT

Auxotrophic microorganisms are often used for genetic engineering, because their biosynthetic deficiency can be complemented by the transforming DNA and allows selection for transformants that have become prototrophic. However, when complementation is obtained by ectopic expression this may lead to unpredictable side effects on the phenotype and, consequently, misinterpretation of experimental data. There are various ways to overcome the problem of auxotrophy, but the most reliable is to restore the function of the defective biosynthetic gene at the native genomic locus. This can be done by either sexual crossing or further genetic engineering. For fungal species lacking a perfect state or situations in which gene targeting is generally cumbersome we have developed a concept that allows transient disruption of
pyrG
. When the gene is in the disrupted state, multiple rounds of gene targeting can be performed with the strain. Once the desired genome engineering is completed,
pyrG
function can be rapidly returned to wild type by a simple selection scheme.
Trypanosoma brucei
RNA Binding Proteins p34 and p37 Mediate NOPP44/46 Cellular Localization via the Exportin 1 Nuclear Export Pathway
Authors: Hellman Kristina , Prohaska Kimberly , Williams Noreen 
Published: October 2007

ABSTRACT

We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to interact with a family of nucleolar phosphoproteins, NOPP44/46, in
Trypanosoma brucei
. These proteins are nearly identical, the major difference being an 18-amino-acid insert in the N terminus of p37. In order to characterize the interaction between p34 and p37 and NOPP44/46, we have utilized an RNA interference (RNAi) cell line that specifically targets p34 and p37. Within these RNAi cells, we detected a disruption of a higher-molecular-weight complex containing NOPP44/46, as well as a dramatic increase in nuclear NOPP44/46 protein levels. We demonstrated that no change occurred in NOPP44/46 mRNA steady-state levels or stability, nor was there a change in cellular protein levels. These results led us to investigate whether p34 and p37 regulate NOPP44/46 cellular localization. Examination of the p34 and p37 amino acid sequences revealed a leucine-rich nuclear export signal, which interacts with the nuclear export factor exportin 1. Immune capture experiments demonstrated that p34, p37, and NOPP44/46 associate with exportin 1. When these experiments were performed with p34/p37 RNAi cells, NOPP44/46 no longer associated with exportin 1. Sequential immune capture experiments demonstrated that p34, p37, NOPP44/46, and exportin 1 exist in a common complex. Inhibiting exportin 1-mediated nuclear export led to an increase in nuclear NOPP44/46 proteins, indicating that they are exported from the nucleus via this pathway. Together, our results demonstrate that p34 and p37 regulate NOPP44/46 cellular localization by facilitating their association with exportin 1.
Endocytosis in the Shiitake Mushroom
Lentinula edodes
and Involvement of GTPase LeRAB7
Authors: Lee Ming Tsung , Szeto Carol Ying Ying , Ng Tak Pan , Kwan Hoi Shan 
Published: October 2007

ABSTRACT

Endocytosis is the process by which substrates enter a cell without passing through the plasma membrane but rather invaginate the cell membrane and form intracellular vesicles.
Rab7
regulates endocytic trafficking between early and late endosomes and between late endosomes and lysosomes. Le
Rab7
in
Lentinula edodes
is strongly homologous to
Rab7
in
Homo sapiens
. Receptors for activated C kinase-1 (LeRACK1) and Rab5 GTPase (LeRAB5) were isolated as interacting partners of LeRab7, and the interactions were confirmed by in vivo and in vitro protein interaction assays. The three genes showed differential expression in the various developmental stages of the mushroom. In situ hybridization showed that the three transcripts were localized in regions of active growth, such as the outer region of trama cells, and the subhymenium of the hymenophore of mature fruiting bodies and the prehymenophore of young fruiting bodies. The existence of endocytosis in the mycelium and hymenophores was confirmed by the internalization of FM4-64. LeRAB7 was partially colocalized with the AM4-64 and was located in the late endocytic pathway. This is the first report of the presence of endocytosis in homobasidiomycetes. LeRAB7, LeRAB5, and LeRACK1 may contribute to the growth of
L. edodes
and cell differentiation in hymenophores.
Unexpected Link between Metal Ion Deficiency and Autophagy in
Aspergillus fumigatus
Authors: Richie Daryl L. , Fuller Kevin K. , Fortwendel Jarrod , Miley Michael D. , McCarthy Jason W. , Feldmesser Marta , Rhodes Judith C. , Askew David S. 
Published: October 2007

ABSTRACT

Autophagy is the major cellular pathway for bulk degradation of cytosolic material and is required to maintain viability under starvation conditions. To determine the contribution of autophagy to starvation stress responses in the filamentous fungus
Aspergillus fumigatus
, we disrupted the
A. fumigatus atg1
gene, encoding a serine/threonine kinase required for autophagy. The ΔAf
atg1
mutant showed abnormal conidiophore development and reduced conidiation, but the defect could be bypassed by increasing the nitrogen content of the medium. When transferred to starvation medium, wild-type hyphae were able to undergo a limited amount of growth, resulting in radial expansion of the colony. In contrast, the ΔAf
atg1
mutant was unable to grow under these conditions. However, supplementation of the medium with metal ions rescued the ability of the ΔAf
atg1
mutant to grow in the absence of a carbon or nitrogen source. Depleting the medium of cations by using EDTA was sufficient to induce autophagy in wild-type
A. fumigatus
, even in the presence of abundant carbon and nitrogen, and the ΔAf
atg1
mutant was severely growth impaired under these conditions. These findings establish a role for autophagy in the recycling of internal nitrogen sources to support conidiophore development and suggest that autophagy also contributes to the recycling of essential metal ions to sustain hyphal growth when exogenous nutrients are scarce.
The
Aspergillus fumigatus
Transcriptional Regulator AfYap1 Represents the Major Regulator for Defense against Reactive Oxygen Intermediates but Is Dispensable for Pathogenicity in an Intranasal Mouse Infection Model
Authors: Lessing Franziska , Kniemeyer Olaf , Wozniok Iwona , Loeffler Juergen , Kurzai Oliver , Haertl Albert , Brakhage Axel A. 
Published: October 2007

ABSTRACT

Macrophages and neutrophils kill the airborne fungal pathogen
Aspergillus fumigatus
. The dependency of this killing process on reactive oxygen intermediates (ROI) has been strongly suggested. Therefore, we investigated the enzymatic ROI detoxifying system by proteome analysis of
A. fumigatus
challenged by H
2
O
2
. Since many of the identified proteins and genes are apparently regulated by a putative
Saccharomyces cerevisiae
Yap1 homolog, the corresponding gene of
A. fumigatus
was identified and designated Af
yap1
. Nuclear localization of a functional AfYap1-eGFP fusion was stress dependent. Deletion of the Af
yap1
gene led to drastically increased sensitivity of the deletion mutant against H
2
O
2
and menadione, but not against diamide and NO radicals. Proteome analysis of the ΔAf
yap1
mutant strain challenged with 2 mM H
2
O
2
indicated that 29 proteins are controlled directly or indirectly by AfYap1, including catalase 2. Despite its importance for defense against reactive agents, the Af
yap1
deletion mutant did not show attenuated virulence in a murine model of
Aspergillus
infection. These data challenge the hypothesis that ROI such as superoxide anions and peroxides play a direct role in killing of
A. fumigatus
in an immunocompromised host. This conclusion was further supported by the finding that killing of
A. fumigatus
wild-type and ΔAf
yap1
mutant germlings by human neutrophilic granulocytes worked equally well irrespective of whether the ROI scavenger glutathione or an NADPH-oxidase inhibitor was added to the cells.
Expression and Characterization of the Flocculin Flo11/Muc1, a
Saccharomyces cerevisiae
Mannoprotein with Homotypic Properties of Adhesion
Authors: Douglas Lois M. , Li Li , Yang Yang , Dranginis A. M. 
Published: October 2007

ABSTRACT

The Flo11/Muc1 flocculin has diverse phenotypic effects.
Saccharomyces cerevisiae
cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that
S. cerevisiae
var.
diastaticus
strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to
S. cerevisiae
var.
diastaticus
cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to
S. cerevisiae
var.
diastaticus
cells expressing
FLO11
and failed to bind to cells with a deletion of
FLO11
, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.
Functional Analysis of the M.HpyAIV DNA Methyltransferase of
Helicobacter pylori
Authors: Skoglund Anna , Björkholm Britta , Nilsson Christina , Andersson Anders F. , Jernberg Cecilia , Schirwitz Katja , Enroth Cristofer , Krabbe Margareta , Engstrand Lars 
Published: October 2007

ABSTRACT

A large number of genes encoding restriction-modification (R-M) systems are found in the genome of the human pathogen
Helicobacter pylori
. R-M genes comprise approximately 10% of the strain-specific genes, but the relevance of having such an abundance of these genes is not clear. The type II methyltransferase (MTase) M.HpyAIV, which recognizes GANTC sites, was present in 60% of the
H. pylori
strains analyzed, whereof 69% were resistant to restriction enzyme digestion, which indicated the presence of an active MTase.
H. pylori
strains with an inactive M.HpyAIV phenotype contained deletions in regions of homopolymers within the gene, which resulted in premature translational stops, suggesting that M.HpyAIV may be subjected to phase variation by a slipped-strand mechanism. An M.HpyAIV gene mutant was constructed by insertional mutagenesis, and this mutant showed the same viability and ability to induce interleukin-8 in epithelial cells as the wild type in vitro but had, as expected, lost the ability to protect its self-DNA from digestion by a cognate restriction enzyme. The M.HpyAIV from
H. pylori
strain 26695 was overexpressed in
Escherichia coli
, and the protein was purified and was able to bind to DNA and protect GANTC sites from digestion in vitro. A bioinformatic analysis of the number of GANTC sites located in predicted regulatory regions of
H. pylori
strains 26695 and J99 resulted in a number of candidate genes.
katA
, a selected candidate gene, was further analyzed by quantitative real-time reverse transcription-PCR and shown to be significantly down-regulated in the M.HpyAIV gene mutant compared to the wild-type strain. This demonstrates the influence of M.HpyAIV methylation in gene expression.
Response of
Desulfovibrio vulgaris
to Alkaline Stress
Authors: Stolyar Sergey , He Qiang , Joachimiak Marcin P. , He Zhili , Yang Zamin Koo , Borglin Sharon E. , Joyner Dominique C. , Huang Katherine , Alm Eric , Hazen Terry C. , Zhou Jizhong , Wall Judy D. , Arkin Adam P. , Stahl David A. 
Published: October 2007

ABSTRACT

The response of exponentially growing
Desulfovibrio vulgaris
Hildenborough to pH 10 stress was studied using oligonucleotide microarrays and a study set of mutants with genes suggested by microarray data to be involved in the alkaline stress response deleted. The data showed that the response of
D. vulgaris
to increased pH is generally similar to that of
Escherichia coli
but is apparently controlled by unique regulatory circuits since the alternative sigma factors (sigma S and sigma E) contributing to this stress response in
E. coli
appear to be absent in
D. vulgaris
. Genes previously reported to be up-regulated in
E. coli
were up-regulated in
D. vulgaris
; these genes included three ATPase genes and a tryptophan synthase gene. Transcription of chaperone and protease genes (encoding ATP-dependent Clp and La proteases and DnaK) was also elevated in
D. vulgaris
. As in
E. coli
, genes involved in flagellum synthesis were down-regulated. The transcriptional data also identified regulators, distinct from sigma S and sigma E, that are likely part of a
D. vulgaris
Hildenborough-specific stress response system. Characterization of a study set of mutants with genes implicated in alkaline stress response deleted confirmed that there was protective involvement of the sodium/proton antiporter NhaC-2, tryptophanase A, and two putative regulators/histidine kinases (DVU0331 and DVU2580).
Characterization of the
Mycobacterium tuberculosis
4-Diphosphocytidyl-2-
C
-Methyl-
d
-Erythritol Synthase: Potential for Drug Development
Authors: Eoh Hyungjin , Brown Amanda C. , Buetow Lori , Hunter William N. , Parish Tanya , Kaur Devinder , Brennan Patrick J. , Crick Dean C. 
Published: October 2007

ABSTRACT

Mycobacterium tuberculosis
utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate and its isomer, dimethylallyl diphosphate, precursors of all isoprenoid compounds. This pathway is of interest as a source of new drug targets, as it is absent from humans and disruption of the responsible genes has shown a lethal phenotype for
Escherichia coli
. In the MEP pathway, 4-diphosphocytidyl-2-
C
-methyl-
d
-erythritol is formed from 2-
C
-methyl-
d
-erythritol 4-phosphate (MEP) and CTP in a reaction catalyzed by a 4-diphosphocytidyl-2-
C
-methyl-
d
-erythritol synthase (IspD). In the present work, we demonstrate that Rv3582c is essential for
M. tuberculosis
: Rv3582c has been cloned and expressed, and the encoded protein has been purified. The purified
M. tuberculosis
IspD protein was capable of catalyzing the formation of 4-diphosphocytidyl-2-
C
-methyl-
d
-erythritol in the presence of MEP and CTP. The enzyme was active over a broad pH range (pH 6.0 to 9.0), with peak activity at pH 8.0. The activity was absolutely dependent upon divalent cations, with 20 mM Mg
2+
being optimal, and replacement of CTP with other nucleotide 5′-triphosphates did not support activity. Under the conditions tested,
M. tuberculosis
IspD had

K
m

values of 58.5 μM for MEP and 53.2 μM for CTP. Calculated
k
cat
and
k
cat
/

K
m

values were 0.72 min
−1
and 12.3 mM
−1
min
−1
for MEP and 1.0 min
−1
and 18.8 mM
−1
min
−1
for CTP, respectively.
Multiple Superoxide Dismutases in
Agrobacterium tumefaciens
: Functional Analysis, Gene Regulation, and Influence on Tumorigenesis
Authors: Saenkham Panatda , Eiamphungporn Warawan , Farrand Stephen K. , Vattanaviboon Paiboon , Mongkolsuk Skorn 
Published: October 2007

ABSTRACT

Agrobacterium tumefaciens
possesses three iron-containing superoxide dismutases (FeSods) encoded by distinct genes with differential expression patterns. SodBI and SodBII are cytoplasmic isozymes, while SodBIII is a periplasmic isozyme.
sodBI
is expressed at a high levels throughout all growth phases.
sodBII
expression is highly induced upon exposure to superoxide anions in a SoxR-dependent manner.
sodBIII
is expressed only during stationary phase. Analysis of the physiological function of
sod
s reveals that the inactivation of
sodBI
markedly reduced levels of resistance to a superoxide generator, menadione. A mutant lacking all three Sod enzymes is the most sensitive to menadione treatment, indicating that all
sod
s contribute at various levels towards the overall menadione resistance level. Sods also have important roles in
A. tumefaciens
virulence toward a host plant. A
sodBI
but not a
sodBII
or
sodBIII
mutant showed marked reduction in its ability to induce tumors on tobacco leaf discs, while the triple
sod
null mutant is avirulent.
Comparative Genomic Hybridization Detects Secondary Chromosomal Deletions in
Escherichia coli
K-12 MG1655 Mutants and Highlights Instability in the
flhDC
Region
Authors: Hobman Jon L. , Patel Mala D. , Hidalgo-Arroyo G. Aida , Cariss S. James L. , Avison Matthew B. , Penn Charles W. , Constantinidou Chrystala 
Published: October 2007

ABSTRACT

The use of whole-genome microarrays for monitoring mutagenized or otherwise engineered genetic derivatives is a potentially powerful tool for checking genomic integrity. Using comparative genomic hybridization of a number of unrelated, directed deletion mutants in
Escherichia coli
K-12 MG1655, we identified unintended secondary genomic deletions in the
flhDC
region in Δ
fnr
, Δ
crp
, and Δ
creB
mutants. These deletions were confirmed by PCR and phenotypic tests. Our findings show that nonmotile progeny are found in some MG1655 directed deletion mutants, and studies on the effects of gene knockouts should be viewed with caution when the mutants have not been screened for the presence of secondary deletions or confirmed by other methods.
A Conserved Glycine Residue of Trimeric Autotransporter Domains Plays a Key Role inYersiniaAdhesin A Autotransport
Authors: Grosskinsky Ulrike , Schütz Monika , Fritz Michaela , Schmid Yvonne , Lamparter Marina C. , Szczesny Pawel , Lupas Andrei N. , Autenrieth Ingo B. , Linke Dirk 
Published: October 2007

ABSTRACTTheYersiniaadhesin A (YadA) is a trimeric autotransporter adhesin of enteric yersiniae. It consists of three major domains: a head mediating adherence to host cells, a stalk involved in serum resistance, and an anchor that forms a membrane pore and is responsible for the autotransport function. The anchor contains a glycine residue, nearly invariant throughout trimeric autotransporter adhesins, that faces the pore lumen. To address the role of this glycine, we replaced it with polar amino acids of increasing side chain size and expressed wild-type and mutant YadA inEscherichia coli. The mutations did not impair the YadA-mediated adhesion to collagen and to host cells or the host cell cytokine production, but they decreased the expression levels and stability of YadA trimers with increasing side chain size. Likewise, autoagglutination and resistance to serum were decreased in these mutants. We found that the periplasmic protease DegP is involved in the degradation of YadA and that in anE. coli degPdeletion strain, mutant versions of YadA were expressed almost to wild-type levels. We conclude that the conserved glycine residue affects both the export and the stability of YadA and consequently some of its putative functions in pathogenesis.
Spatial Organization ofMyxococcus xanthusduring Fruiting Body Formation
Authors: Curtis Patrick D. , Taylor Rion G. , Welch Roy D. , Shimkets Lawrence J. 
Published: October 2007

ABSTRACTMicrocinematography was used to examine fruiting body development ofMyxococcus xanthus. Wild-type cells progress through three distinct phases: a quiescent phase with some motility but little aggregation (0 to 8 h), a period of vigorous motility leading to raised fruiting bodies (8 to 16 h), and a period of maturation during which sporulation is initiated (16 to 48 h). Fruiting bodies are extended vertically in a series of tiers, each involving the addition of a cell monolayer on top of the uppermost layer. ApilA(MXAN_5783) mutant produced less extracellular matrix material and thus allowed closer examination of tiered aggregate formation. AcsgA(MXAN_1294) mutant exhibited no quiescent phase, aberrant aggregation in phase 2, and disintegration of the fruiting bodies in the third phase.
Identification and Characterization of the Flavin:NADH Reductase (PrnF) Involved in a Novel Two-Component Arylamine Oxygenase
Authors: Lee Jung-Kul , Zhao Huimin 
Published: October 2007

ABSTRACT

Two-component oxygenases catalyze a wide variety of important oxidation reactions. Recently we characterized a novel arylamine N-oxygenase (PrnD), a new member of the two-component oxygenase family (J. Lee et al., J. Biol. Chem.
280
:36719-36728, 2005). Although arylamine N-oxygenases are widespread in nature, aminopyrrolnitrin
N
-oxygenase (PrnD) represents the only biochemically and mechanistically characterized arylamine N-oxygenase to date. Here we report the use of bioinformatic and biochemical tools to identify and characterize the reductase component (PrnF) involved in the PrnD-catalyzed unusual arylamine oxidation. The
prnF
gene was identified via sequence analysis of the whole genome of
Pseudomonas fluorescens
Pf-5 and subsequently cloned and overexpressed in
Escherichia coli
. The purified PrnF protein catalyzes reduction of flavin adenine dinucleotide (FAD) by NADH with a
k
cat
of 65 s
−1
(
K

m

= 3.2 μM for FAD and 43.1 μM for NADH) and supplies reduced FAD to the PrnD oxygenase component. Unlike other known reductases in two-component oxygenase systems, PrnF strictly requires NADH as an electron donor to reduce FAD and requires unusual protein-protein interaction with the PrnD component for the efficient transfer of reduced FAD. This PrnF enzyme represents the first cloned and characterized flavin reductase component in a novel two-component arylamine oxygenase system.
Control of the Transcription of a Short Gene Encoding a Cyclic Peptide in
Streptococcus thermophilus
: a New Quorum-Sensing System?
Authors: Ibrahim Mariam , Guillot Alain , Wessner Francoise , Algaron Florence , Besset Colette , Courtin Pascal , Gardan Rozenn , Monnet Véronique 
Published: October 2007

ABSTRACT

Gram-positive bacteria secrete a variety of peptides that are often subjected to posttranslational modifications and that are either antimicrobials or pheromones involved in bacterial communication. Our objective was to identify peptides secreted by
Streptococcus thermophilus
, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria, and to understand their potential roles in cell-cell communication. Using reverse-phase liquid chromatography, mass spectrometry, and Edman sequencing, we analyzed the culture supernatants of three
S. thermophilus
strains (CNRZ1066, LMG18311, and LMD-9) grown in a medium containing no peptides. We identified several peptides in the culture supernatants, some of them found with the three strains while others were specific to the LMD-9 strain. We focused our study on a new modified peptide secreted by
S. thermophilus
LMD-9 and designated Pep1357C. This peptide contains 9 amino acids and lost 2 Da in a posttranslational modification, most probably a dehydrogenation, leading to a linkage between the Lys2 and Trp6 residues. Production of Pep1357C and transcription of its encoding gene depend on both the medium composition and the growth phase. Furthermore, we demonstrated that transcription of the gene coding for Pep1357C is drastically decreased in mutants inactivated for the synthesis of a short hydrophobic peptide, a transcriptional regulator, or the oligopeptide transport system. Taken together, our results led us to deduce that the transcription of the Pep1357C-encoding gene is controlled by a new quorum-sensing system.