bluuebunny/crossref_metadata_2025_split huggingface dataset:
Acetamide Selection of
Kluyveromyces lactis
Cells Transformed with an Integrative Vector Leads to High-Frequency Formation of Multicopy Strains
Authors: Read Jeremiah D. , Colussi Paul A. , Ganatra Mehul B. , Taron Christopher H. 
Published: June 2007

ABSTRACT

The yeast
Kluyveromyces lactis
has been extensively used as a host for heterologous protein expression. A necessary step in the construction of a stable expression strain is the introduction of an integrative expression vector into
K. lactis
cells, followed by selection of transformed strains using either medium containing antibiotic (e.g., G418) or nitrogen-free medium containing acetamide. In this study, we show that selection using acetamide yields
K. lactis
transformant populations nearly completely comprised of strains bearing multiple tandem insertions of the expression vector pKLAC1 at the
LAC4
chromosomal locus, whereas an average of 16% of G418-selected transformants are multiply integrated. Additionally, the average copy number within transformant populations doubled when acetamide was used for selection compared to G418. Finally, we demonstrate that the high frequency of multicopy integration associated with using acetamide selection can be exploited to rapidly construct expression strains that simultaneously produce multiple heterologous proteins or multisubunit proteins, such as Fab antibodies.
Use of Propidium Monoazide for Live/Dead Distinction in Microbial Ecology
Authors: Nocker Andreas , Sossa-Fernandez Priscilla , Burr Mark D. , Camper Anne K. 
Published: June 2007

ABSTRACT
One of the prerequisites of making ecological conclusions derived from genetic fingerprints is that bacterial community profiles reflect the live portion of the sample of interest. Propidium monoazide is a membrane-impermeant dye that selectively penetrates cells with compromised membranes, which can be considered dead. Once inside the cells, PMA intercalates into the DNA and can be covalently cross-linked to it, which strongly inhibits PCR amplification. By using PCR after PMA treatment, the analysis of bacterial communities can theoretically be limited to cells with intact cell membranes. Four experiments were performed to study the usefulness of PMA treatment of mixed bacterial communities comprising both intact and compromised cells in combination with end-point PCR by generating community profiles from the following samples: (i) defined mixtures of live and isopropanol-killed cells from pure cultures of random environmental isolates, (ii) wastewater treatment plant influent spiked with defined ratios of live and dead cells, (iii) selected environmental communities, and (iv) a water sediment sample exposed to increasing heat stress. Regions of 16S rRNA genes were PCR amplified from extracted genomic DNA, and PCR products were analyzed by using denaturing gradient gel electrophoresis (DGGE). Results from the first two experiments show that PMA treatment can be of value with end-point PCR by suppressing amplification of DNA from killed cells. The last two experiments suggest that PMA treatment can affect banding patterns in DGGE community profiles and their intensities, although the intrinsic limitations of end-point PCR have to be taken into consideration.
EstB-Mediated Hydrolysis of the Siderophore Triacetylfusarinine C Optimizes Iron Uptake of
Aspergillus fumigatus
Authors: Kragl Claudia , Schrettl Markus , Abt Beate , Sarg Bettina , Lindner Herbert H. , Haas Hubertus 
Published: June 2007

ABSTRACT

Aspergillus fumigatus
excretes the fusarinine-type siderophore desferri-triacetylfusarinine C (DF-TafC) to mobilize iron. DF-TafC is a cyclic peptide consisting of three
N

5

-
cis
-anhydromevalonyl-
N

5

-hydroxy-
N

2

-acetyl-
l
-ornithine residues linked by ester bonds; these linkages are in contrast to peptide linkages found for ferrichrome-type siderophores. Subsequent to the binding of iron and uptake, triacetylfusarinine C (TafC) is hydrolyzed, the cleavage products are excreted, and the iron is transferred to the metabolism or to the intracellular siderophore desferri-ferricrocin (DF-FC) for iron storage. Here we report the identification and characterization of the TafC esterase EstB, the first eukaryotic siderophore-degrading enzyme to be characterized at the molecular level. The encoding gene,
estB
, was found to be located in an iron-regulated gene cluster, indicating a role in iron metabolism. Deletion of
estB
in
A. fumigatus
eliminated TafC esterase activity of cellular extracts and caused increased intracellular accumulation of TafC and TafC hydrolysis products in vivo.
Escherichia coli
-expressed EstB displayed specific TafC esterase activity but did not hydrolyze fusarinine C, which has the same core structure as TafC but lacks three
N

2

-acetyl residues. Localization of EstB via enhanced green fluorescent protein tagging suggested that TafC hydrolysis takes place in the cytoplasm. EstB abrogation reduced the intracellular transfer rate of iron from TafC to DF-FC and delayed iron sensing. Furthermore, EstB deficiency caused a decreased radial growth rate under iron-depleted but not iron-replete conditions. Taken together, these data suggest that EstB-mediated TafC hydrolysis optimizes but is not essential for TafC-mediated iron uptake in
A. fumigatus
.
Conservation of PEX19-Binding Motifs Required for Protein Targeting to Mammalian Peroxisomal and Trypanosome Glycosomal Membranes
Authors: Saveria Tracy , Halbach André , Erdmann Ralf , Volkmer-Engert Rudolf , Landgraf Christiane , Rottensteiner Hanspeter , Parsons Marilyn 
Published: June 2007

ABSTRACT

Glycosomes are divergent peroxisomes found in trypanosomatid protozoa, including those that cause severe human diseases throughout much of the world. While peroxisomes are dispensable for both yeast (
Saccharomyces cerevisiae
and others) and mammalian cells in vitro, glycosomes are essential for trypanosomes and hence are viewed as a potential drug target. The import of proteins into the matrix of peroxisomes utilizes multiple peroxisomal membrane proteins which require the peroxin PEX19 for insertion into the peroxisomal membrane. In this report, we show that the specificity of peroxisomal membrane protein binding for
Trypanosoma brucei
PEX19 is very similar to those previously identified for human and yeast PEX19. Our studies show that trafficking is conserved across these distant phyla and that both a PEX19 binding site and a transmembrane domain are required for the insertion of two test proteins into the glycosomal membrane. However, in contrast to
T. brucei
PEX10 and PEX12,
T. brucei
PEX14 does not traffic to human peroxisomes, indicating that it is not recognized by the human PEX14 import mechanism.
Role of Heat Shock Transcription Factor in
Saccharomyces cerevisiae
Oxidative Stress Response
Authors: Yamamoto Ayako , Ueda Junko , Yamamoto Noritaka , Hashikawa Naoya , Sakurai Hiroshi 
Published: June 2007

ABSTRACT

The heat shock transcription factor Hsf1 of the yeast
Saccharomyces cerevisiae
regulates the transcription of a set of genes that contain heat shock elements (HSEs) in their promoters and function in diverse cellular processes, including protein folding. Here, we show that Hsf1 activates the transcription of various target genes when cells are treated with oxidizing reagents, including the superoxide anion generators menadione and KO
2
and the thiol oxidants diamide and 1-chloro-2,4-dinitrobenzene (CDNB). Similar to heat shock, the oxidizing reagents are potent inducers of both efficient HSE binding and extensive phosphorylation of Hsf1. The inducible phosphorylation of Hsf1 is regulated by the intramolecular domain-domain interactions and affects HSE structure-specific transcription. Unlike the heat shock, diamide, or CDNB response, menadione or KO
2
activation of Hsf1 is inhibited by cyclic-AMP-dependent protein kinase (PKA) activity, which negatively regulates the activator functions of other transcriptional regulators implicated in the oxidative stress response. These results demonstrate that Hsf1 is a member of the oxidative stress-responsive activators and that PKA is a general negative regulator in the superoxide anion response.
Development of a Gene Knockout System in
Candida parapsilosis
Reveals a Conserved Role for
BCR1
in Biofilm Formation
Authors: Ding Chen , Butler Geraldine 
Published: June 2007

ABSTRACT

Candida parapsilosis
is an important cause of candidiasis, yet few molecular tools are available. We adapted a recyclable nourseothricin resistance marker gene (
SAT1
) originally developed for use with
C. albicans
in order to generate gene knockouts from
C. parapsilosis
. We first replaced the promoters driving expression of the FLP recombinase and the
SAT1
genes with the equivalent sequences from
C. parapsilosis
. We then used the cassette to generate a homozygous knockout of
C. parapsilosis URA3
. The
ura3
knockouts have altered colony morphologies. We also knocked out both alleles of an ortholog of
BCR1
, a gene encoding a transcription factor known to be required for biofilm development in
C. albicans
. We show that
C. parapsilosis BCR1
is necessary for biofilm development in
C. parapsilosis
and for expression of the cell wall protein encoded by
RBT1
. Our results suggest that there are significant similarities in the regulation of biofilms between the two species, despite the fact that
C. parapsilosis
does not generate true hyphae and that
BCR1
regulates the expression of many hypha-specific adhesins in
C. albicans
.
Lia1p, a Novel Protein Required during Nuclear Differentiation for Genome-Wide DNA Rearrangements in
Tetrahymena thermophila
Authors: Rexer Charles H. , Chalker Douglas L. 
Published: June 2007

ABSTRACT

Extensive genome-wide rearrangements occur during somatic macronuclear development in
Tetrahymena thermophila
. These events are guided by RNA interference-directed chromatin modification including histone H3 lysine 9 methylation, which marks specific germ line-limited internal eliminated sequences (IESs) for excision. Several genes putatively involved in these developmental genome rearrangements were identified based on their proteins' localization to differentiating somatic nuclei, and here we demonstrate that one,
LIA1
, encodes a novel protein that is an essential component of the genome rearrangement machinery. A green fluorescent protein-Lia1 fusion protein exhibited dynamic nuclear localization during development that has striking similarity to that of the dual chromodomain-containing DNA rearrangement protein, Pdd1p. Coimmunoprecipitation experiments showed that Lia1p associates with Pdd1p and IES chromatin during macronuclear development. Cell lines in which we disrupted both the germ line and somatic copies of
LIA1
(Δ
LIA1
) grew normally but were unable to generate viable progeny, arresting late in development just prior to returning to vegetative growth. These mutant lines failed to properly form Pdd1p-containing nuclear structures and eliminate IESs despite showing normal levels of H3K9 methylation. These data indicate that Lia1p is required late in conjugation for the reorganization of the
Tetrahymena
genome.
GroEL Plays a Central Role in Stress-Induced Negative Regulation of Bacterial Conjugation by Promoting Proteolytic Degradation of the Activator Protein TraJ
Authors: Zahrl Doris , Wagner Andrea , Tscherner Michael , Koraimann Günther 
Published: June 2007

ABSTRACT

Transcription of DNA transfer genes is a prerequisite for conjugative DNA transfer of F-like plasmids. Transfer gene expression is sensed by the donor cell and is regulated by a complex network of plasmid- and host-encoded factors. In this study we analyzed the effect of induction of the heat shock regulon on transfer gene expression and DNA transfer in
Escherichia coli
. Raising the growth temperature from 22°C to 43°C transiently reduced transfer gene expression to undetectable levels and reduced conjugative transfer by 2 to 3 orders of magnitude. In contrast, when host cells carried the temperature-sensitive
groEL44
allele, heat shock-mediated repression was alleviated. These data implied that the chaperonin GroEL was involved in negative regulation after heat shock. Investigation of the role of GroEL in this regulatory process revealed that, in
groEL
(Ts) cells, TraJ, the plasmid-encoded master activator of type IV secretion (T4S) system genes, was less susceptible to proteolysis and had a prolonged half-life compared to isogenic wild-type
E. coli
cells. This result suggested a direct role for GroEL in proteolysis of TraJ, down-regulation of T4S system gene expression, and conjugation after heat shock. Strong support for this novel role for GroEL in regulation of bacterial conjugation was the finding that GroEL specifically interacted with TraJ in vivo. Our results further suggested that in wild-type cells this interaction was followed by rapid degradation of TraJ whereas in
groEL
(Ts) cells TraJ remained trapped in the temperature-sensitive GroEL protein and thus was not amenable to proteolysis.
A Large Gene Cluster in
Burkholderia xenovorans
Encoding Abietane Diterpenoid Catabolism
Authors: Smith Daryl J. , Park Joonhong , Tiedje James M. , Mohn William W. 
Published: June 2007

ABSTRACT

Abietane diterpenoids are defense compounds synthesized by trees that are abundant in natural environments and occur as significant pollutants from pulp and paper production.
Burkholderia xenovorans
LB400 has diverse catabolic capabilities and represents an important group of heterotrophic bacteria in soil environments. The genome sequence of LB400 revealed homologs of the
dit
genes of
Pseudomonas abietaniphila
BKME-9, which encode abietane diterpenoid degradation. LB400 grew on abietic acid (AbA), dehydroabietic acid (DhA), palustric acid (PaA), and 7-oxo-DhA. A Xeotron microarray set, with probes for 8450 of the estimated 9000 LB400 genes, was used to compare the transcriptomes of LB400 growing on DhA versus on succinate. On DhA, 97 genes were upregulated, 43 of which were within an 80-kb cluster located on the 1.47-Mbp megaplasmid of LB400. Upregulated genes in this cluster encode a permease, a ring-hydroxylating dioxygenase system (DitA), a ring-cleavage dioxygenase (DitC), a P450 monooxygenase (DitQ), and enzymes catalyzing beta-oxidation-type reactions. Disruption of the
ditA1
gene, encoding the alpha-subunit of DitA, abolished growth on these abietanes. Analyses of the metabolism of abietanes by cell suspensions of wild-type LB400 and the
ditA1
mutant indicate a convergent pathway, with 7-oxo-DhA as a common intermediate for ring hydroxylation by DitA. Also, 7-oxo-PaA was identified as a metabolite of both AbA and PaA. Sequence analysis indicates that genes encoding this pathway have been horizontally transferred among
Betaproteobacteria
and
Gammaproteobacteria
.
Two-Component Response Regulators ofVibrio fischeri: Identification, Mutagenesis, and Characterization
Authors: Hussa Elizabeth A. , O'Shea Therese M. , Darnell Cynthia L. , Ruby Edward G. , Visick Karen L. 
Published: June 2007

ABSTRACTTwo-component signal transduction systems are utilized by prokaryotic and eukaryotic cells to sense and respond to environmental stimuli, both to maintain homeostasis and to rapidly adapt to changing conditions. Studies have begun to emerge that utilize a large-scale mutagenesis approach to analyzing these systems in prokaryotic organisms. Due to the recent availability of its genome sequence, such a global approach is now possible for the marine bioluminescent bacteriumVibrio fischeri, which exists either in a free-living state or as a mutualistic symbiont within a host organism such as the Hawaiian squid speciesEuprymna scolopes. In this work, we identified 40 putative two-component response regulators encoded within theV. fischerigenome. Based on the type of effector domain present, we classified six as NarL type, 13 as OmpR type, and six as NtrC type; the remaining 15 lacked a predicted DNA-binding domain. We subsequently mutated 35 of these genes via a vector integration approach and analyzed the resulting mutants for roles in bioluminescence, motility, and competitive colonization of squid. Through these assays, we identified three novel regulators ofV. fischeriluminescence and seven regulators that altered motility. Furthermore, we found 11 regulators with a previously undescribed effect on competitive colonization of the host squid. Interestingly, five of the newly characterized regulators each affected two or more of the phenotypes examined, strongly suggesting interconnectivity among systems. This work represents the first large-scale mutagenesis of a class of genes inV. fischeriusing a genomic approach and emphasizes the importance of two-component signal transduction in bacterium-host interactions.
Ler and H-NS, Regulators Controlling Expression of the Long Polar Fimbriae of
Escherichia coli
O157:H7
Authors: Torres Alfredo G. , López-Sánchez Guillermo N. , Milflores-Flores Lorena , Patel Shilpa D. , Rojas-López Maricarmen , Martínez de la Peña Claudia F. , Arenas-Hernández Margarita M. P. , Martínez-Laguna Ygnacio 
Published: June 2007

ABSTRACT

Enterohemorrhagic
Escherichia coli
(EHEC) serotype O157:H7 colonizes the human intestine and is responsible for diarrheal outbreaks worldwide. Previously we showed that EHEC produces long polar fimbriae (LPF) and that maximum expression is observed during the exponential phase of growth at 37°C and pH 6.5. In this study, we analyzed the roles of several regulators in the expression of LPF using the β-galactosidase reporter system, and we found that H-NS functions as a transcriptional silencer while Ler functions as an antisilencer of LPF expression. Interestingly, deletion of the
hns
and
ler
genes in EHEC caused constitutive expression of the fusion reporter protein. Semiquantitative reverse transcription (RT)-PCR was also used to analyze LPF expression in the EHEC
ler
or
hns
mutant strain. The
hns
mutant exhibited an increase in
lpf
mRNA expression, while expression in the
ler
mutant was decreased, compared to that in the wild-type strain. Using primer extension analysis, we identified two potential transcriptional start sites within the regulatory region of
lpf
and located consensus hexamers of −10 (CAAGAT) and −35 (TTCAAA), which are commonly found in σ
70
-dependent promoters. Further, we determined whether H-NS and Ler interact directly with the
lpf
promoter region by using purified His-tagged proteins and electrophoretic mobility shift assays. Our data are the first to show direct binding interactions between the H-NS and Ler proteins within the regulatory sequence of the
lpf
operon. Based on the electrophoretic mobility shift assay, RT-PCR, primer extension, and β-galactosidase assay results, we concluded that the
E. coli
O157:H7
lpf
operon possesses a promoter dependent on σ
70
, that H-NS binds to the regulatory sequence of
lpfA
and “silences” the transcription of
lpf
, and that Ler binds to the regulatory sequence and inhibits the action of the H-NS protein.
The Quorum-Sensing Molecule Autoinducer 2 Regulates Motility and Flagellar Morphogenesis in
Helicobacter pylori
Authors: Rader Bethany A. , Campagna Shawn R. , Semmelhack Martin F. , Bassler Bonnie L. , Guillemin Karen 
Published: June 2007

ABSTRACT

The genome of the gastric pathogen
Helicobacter pylori
contains a homologue of the gene
luxS
, which has been shown to be responsible for production of the quorum-sensing signal autoinducer 2 (AI-2). We report here that deletion of the
luxS
gene in strain G27 resulted in decreased motility on soft agar plates, a defect that was complemented by a wild-type copy of the
luxS
gene and by the addition of cell-free supernatant containing AI-2. The flagella of the
luxS
mutant appeared normal; however, in genetic backgrounds lacking any of three flagellar regulators—the two-component sensor kinase
flgS
, the sigma factor σ
28
(also called
fliA
), and the anti-sigma factor
flgM
—loss of
luxS
altered flagellar morphology. In all cases, the double mutant phenotypes were restored to the
luxS
+
phenotype by the addition of synthetic 4,5-dihydroxy-2,3-pentanedione (DPD), which cyclizes to form AI-2. Furthermore, in all mutant backgrounds loss of
luxS
caused a decrease in transcript levels of the flagellar regulator
flhA
. Addition of DPD to
luxS
cells induced
flhA
transcription in a dose-dependent manner. Deletion of
flhA
in a wild-type or
luxS
mutant background resulted in identical loss of motility, flagella, and flagellar gene expression. These data demonstrate that AI-2 functions as a secreted signaling molecule upstream of FlhA and plays a critical role in global regulation of flagellar gene transcription in
H. pylori
.
UnstableEscherichia coliL Forms Revisited: Growth Requires Peptidoglycan Synthesis
Authors: Joseleau-Petit Danièle , Liébart Jean-Claude , Ayala Juan A. , D'Ari Richard 
Published: June 2007

ABSTRACTGrowing bacterial L forms are reputed to lack peptidoglycan, although cell division is normally inseparable from septal peptidoglycan synthesis. To explore which cell division functions L forms use, we established a protocol for quantitatively converting a culture of a wild-typeEscherichia coliK-12 strain overnight to a growing L-form-like state by use of the β-lactam cefsulodin, a specific inhibitor of penicillin-binding proteins (PBPs) 1A and 1B. In rich hypertonic medium containing cefsulodin, all cells are spherical and osmosensitive, like classical L forms. Surprisingly, however, mutant studies showed that colony formation requiresd-glutamate, diaminopimelate, and MurA activity, all of which are specific to peptidoglycan synthesis. High-performance liquid chromatography analysis confirmed that these L-form-like cells contain peptidoglycan, with 7% of the normal amount. Moreover, the β-lactam piperacillin, a specific inhibitor of the cell division protein PBP 3, rapidly blocks the cell division of these L-form-like cells. Similarly, penicillin-induced L-form-like cells, which grow only within the agar layers of rich hypertonic plates, also required-glutamate, diaminopimelate, and MurA activity. These results strongly suggest that cefsulodin- and penicillin-induced L-form-like cells ofE. coli—and possibly all L forms—have residual peptidoglycan synthesis which is essential for their growth, probably being required for cell division.
BigR, a Transcriptional Repressor from Plant-Associated Bacteria, Regulates an Operon Implicated in Biofilm Growth
Authors: Barbosa Rosicler L. , Benedetti Celso E. 
Published: June 2007

ABSTRACT

Xylella fastidiosa
is a plant pathogen that colonizes the xylem vessels, causing vascular occlusion due to bacterial biofilm growth. However, little is known about the molecular mechanisms driving biofilm formation in
Xylella
-plant interactions. Here we show that BigR (for “biofilm growth-associated repressor”) is a novel helix-turn-helix repressor that controls the transcription of an operon implicated in biofilm growth. This operon, which encodes BigR, membrane proteins, and an unusual beta-lactamase-like hydrolase (BLH), is restricted to a few plant-associated bacteria, and thus, we sought to understand its regulation and function in
X. fastidiosa
and
Agrobacterium tumefaciens
. BigR binds to a palindromic AT-rich element (the BigR box) in the
Xylella
and
Agrobacterium blh
promoters and strongly represses the transcription of the operon in these cells. The BigR box overlaps with two alternative −10 regions identified in the
blh
promoters, and mutations in this box significantly affected transcription, indicating that BigR competes with the RNA polymerase for the same promoter site. Although BigR is similar to members of the ArsR/SmtB family of regulators, our data suggest that, in contrast to the initial prediction, it does not act as a metal sensor. Increased activity of the BigR operon was observed in both
Xylella
and
Agrobacterium
biofilms. In addition, an
A. tumefaciens bigR
mutant showed constitutive expression of operon genes and increased biofilm formation on glass surfaces and tobacco roots, indicating that the operon may play a role in cell adherence or biofilm development.