bluuebunny/crossref_metadata_2025_split huggingface dataset:
High-Frequency Phage-Mediated Gene Transfer among
Escherichia coli
Cells, Determined at the Single-Cell Level
Authors: Kenzaka Takehiko , Tani Katsuji , Sakotani Akiko , Yamaguchi Nobuyasu , Nasu Masao 
Published: March 2007

ABSTRACT

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among
Escherichia coli
cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated
E. coli
phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3 × 10
−8
to 2 × 10
−6
, 1 × 10
−8
to 4 × 10
−8
, and <4 × 10
−9
to 4 × 10
−8
per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7 × 10
−4
to 1 × 10
−3
, 9 × 10
−4
to 3 × 10
−3
, and 5 × 10
−4
to 4 × 10
−3
for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.
S-Layer-Mediated Display of the Immunoglobulin G-Binding Domain of Streptococcal Protein G on the Surface ofCaulobacter crescentus: Development of an Immunoactive Reagent
Authors: Nomellini John F. , Duncan Gillian , Dorocicz Irene R. , Smit John 
Published: March 2007

ABSTRACTThe immunoglobulin G (IgG)-binding streptococcal protein G is often used for immunoprecipitation or immunoadsorption-based assays, as it exhibits binding to a broader spectrum of host species IgG and IgG subclasses than the alternative,Staphylococcus aureusprotein A.Caulobacter crescentusproduces a hexagonally arranged paracrystalline protein surface layer (S-layer) composed of a single secreted protein, RsaA, that is notably tolerant of heterologous peptide insertions while maintaining the surface-attached crystalline character. Here, a protein G IgG-binding domain, GB1, was expressed as an insertion into full-length RsaA on the cell surface to produce densely packed immunoreactive particles. GB1 insertions at five separate sites were expressed, and all bound rabbit and goat IgG, but expression levels were reduced compared to those of wild-type RsaA and poor binding to mouse IgG was noted. To remedy this, we used the 20-amino-acid Muc1 peptide derived from human mucins as a spacer, since insertions of multiple tandem repeats were well tolerated for RsaA secretion and assembly. This strategy worked remarkably well, and recombinant RsaA proteins, containing up to three GB1 domains, surrounded by Muc1 peptides, not only were secreted and assembled but did so at wild-type levels. The ability to bind IgG (including mouse IgG) increased as GB1 units were added, and those with three GB1 domains bound twice as much rabbit IgG per cell asS. aureuscells (Pansorbin). The ability of recombinant protein G-Caulobactercells to function as immunoactive reagents was assessed in an immunoprecipitation assay using a FLAG-tagged protein and anti-FLAG mouse monoclonal antibody; their performance was comparable to that of protein G-Sepharose beads. This work demonstrates the potential for using cells expressing recombinant RsaA/GB1 in immunoassays, especially considering that protein G-Caulobactercells are more cost-effective than protein G beads and exhibit a broader species and IgG isotype binding range than protein A.
Increased Production of Xylanase by Expression of a Truncated Version of the
xyn11A
Gene from
Nonomuraea flexuosa
in
Trichoderma reesei
Authors: Paloheimo Marja , Mäntylä Arja , Kallio Jarno , Puranen Terhi , Suominen Pirkko 
Published: March 2007

ABSTRACT

We have previously shown that the
Nonomuraea flexuosa
Xyn11A polypeptides devoid of the carbohydrate binding module (CBM) have better thermostability than the full-length xylanase and are effective in bleaching of pulp. To produce an enzyme preparation useful for industrial applications requiring high temperature, the region encoding the CBM was deleted from the
N. flexuosa xyn11A
gene and the truncated gene was expressed in
Trichoderma reesei
. The xylanase sequence was fused to the
T. reesei
mannanase I (Man5A) signal sequence or 3′ to a
T. reesei
carrier polypeptide, either the Man5A core/hinge or the cellulose binding domain (CBD) of cellobiohydrolase II (Cel6A, CBHII). The gene and fusion genes were expressed using the cellobiohydrolase 1 (
cel7A
,
cbh1
) promoter. Single-copy isogenic transformants in which the expression cassette replaced the
cel7A
gene were cultivated and analyzed. The transformants expressing the truncated
N. flexuosa xyn11A
produced clearly increased amounts of both the xylanase/fusion mRNA and xylanase activity compared to the corresponding strains expressing the full-length
N. flexuosa xyn11A
. The transformant expressing the
cel6A
CBD-truncated
N. flexuosa xyn11A
produced about 1.9 g liter
−1
of the xylanase in laboratory-scale fermentations. The xylanase constituted about 25% of the secreted proteins. The production of the truncated xylanase did not induce the unfolded protein response (UPR) pathway. However, the UPR was induced when the full-length
N. flexuosa xyn11A
with an exact fusion to the
cel7A
terminator was expressed. We suggest that the
T. reesei
folding/secretion machinery is not able to cope properly with the bacterial CBM when the mRNA of the full-length
N. flexuosa xyn11A
is efficiently translated.
Crm1-Mediated Nuclear Export of the
Schizosaccharomyces pombe
Transcription Factor Cuf1 during a Shift from Low to High Copper Concentrations
Authors: Beaudoin Jude , Labbé Simon 
Published: March 2007

ABSTRACT

In this study, we examine the fate of the nuclear pool of the
Schizosaccharomyces pombe
transcription factor Cuf1 in response to variations in copper levels. A nuclear pool of Cuf1-green fluorescent protein (GFP) was generated by expressing a functional
cuf1

+

-
GFP
allele in the presence of a copper chelator. We then extinguished
cuf1

+

-
GFP
expression and tracked the changes in the localization of the nuclear pool of Cuf1-GFP in the presence of low or high copper concentrations. Treating cells with copper as well as silver ions resulted in the nuclear export of Cuf1. We identified a leucine-rich nuclear export signal (NES),
349
LAALNHISAL
358
, within the C-terminal region of Cuf1. Mutations in this sequence abrogated Cuf1 export from the nucleus. Furthermore, amino acid substitutions that impair Cuf1 NES function resulted in increased target gene expression and a concomitant cellular hypersensitivity to copper. Export of the wild-type Cuf1 protein was inhibited by leptomycin B (LMB), a specific inhibitor of the nuclear export protein Crm1. We further show that cells expressing a temperature-sensitive mutation in
crm1

+

exhibit increased nuclear accumulation of Cuf1 at the nonpermissive temperature. Although wild-type Cuf1 is localized in the nucleus in both conditions, we observed that the protein can still be inactivated by copper, resulting in the repression of
ctr4

+

gene expression in the presence of exogenous copper. These results demonstrate that nuclear accumulation of Cuf1 per se is not sufficient to cause the unregulated expression of the copper transport genes like
ctr4

+

. In addition to nuclear localization, a functional Cys-rich domain or NES element in Cuf1 is required to appropriately regulate copper transport gene expression in response to changes in intracellular copper concentration.
DNA Sequence Characterization and Molecular Evolution of
MAT1
and
MAT2
Mating-Type Loci of the Self-Compatible Ascomycete Mold
Neosartorya fischeri
Authors: Rydholm C. , Dyer P. S. , Lutzoni F. 
Published: March 2007

ABSTRACT

Degenerate PCR and chromosome-walking approaches were used to identify mating-type (
MAT
) genes and flanking regions from the homothallic (sexually self-fertile) euascomycete fungus
Neosartorya fischeri
, a close relative of the opportunistic human pathogen
Aspergillus fumigatus
. Both putative alpha- and high-mobility-group-domain
MAT
genes were found within the same genome, providing a functional explanation for self-fertility. However, unlike those in many homothallic euascomycetes (Pezizomycotina), the genes were not found adjacent to each other and were termed
MAT1
and
MAT2
to recognize the presence of distinct loci. Complete copies of putative
APN1
(DNA lyase) and
SLA2
(cytoskeleton assembly control) genes were found bordering the
MAT1
locus. Partial copies of
APN1
and
SLA2
were also found bordering the
MAT2
locus, but these copies bore the genetic hallmarks of pseudogenes. Genome comparisons revealed synteny over at least 23,300 bp between the
N. fischeri MAT1
region and the
A. fumigatus MAT
locus region, but no such long-range conservation in the
N. fischeri MAT2
region was evident. The sequence upstream of
MAT2
contained numerous candidate transposase genes. These results demonstrate a novel means involving the segmental translocation of a chromosomal region by which the ability to undergo self-fertilization may be acquired. The results are also discussed in relation to their significance in indicating that heterothallism may be ancestral within the
Aspergillus
section
Fumigati
.
l-Arginine and Cationic Amino Acid Transporter 2B Regulate Growth and Survival ofLeishmania amazonensisAmastigotes in Macrophages
Authors: Wanasen Nanchaya , MacLeod Carol L. , Ellies Lesley G. , Soong Lynn 
Published: March 2007

ABSTRACTLeishmaniaspp. are obligate intracellular parasites, requiring a suitable microenvironment for their growth within host cells. We previously reported that the growth ofLeishmania amazonensisamastigotes in murine macrophages (Mφs) was enhanced in the presence of gamma interferon (IFN-γ), a Th1 cytokine normally associated with classical Mφ activation and killing of intracellular pathogens. In this study, we provided several lines of evidence suggesting that IFN-γ-mediated parasite growth enhancement was associated withl-arginine transport via mouse cationic amino acid transporter 2B (mCAT-2B). (i) mRNA expression ofSlc7A2, the gene encoding for mCAT-2B, as well asl-arginine transport was increased in IFN-γ-treated Mφs. (ii) Supplementation ofl-arginine in Mφ cultures increased parasite growth. (iii) Parasite growth enhancement in wild-type Mφs was inhibited in the presence of nonmetabolizedl-arginine analogues. (iv) IFN-γ-mediated parasite growth was absent in Mφs derived from mCAT-2B-deficient mice. Although we detected a clear upregulation of mCAT-2B andl-arginine transport, no measurable iNOS or arginase activities were observed in IFN-γ-treated, infected Mφs. Together, these data suggest an involvement of a novell-arginine usage independent of iNOS and arginase activities during IFN-γ-mediated parasite growth enhancement. A possible role of mCAT-2B in supplyingl-arginine directly to the parasites for their proliferation is discussed.
Fatty Acids Isolated from
Toxoplasma gondii
Reduce Glycosylphosphatidylinositol-Induced Tumor Necrosis Factor Alpha Production through Inhibition of the NF-κB Signaling Pathway
Authors: Debierre-Grockiego Françoise , Rabi Khamran , Schmidt Jörg , Geyer Hildegard , Geyer Rudolf , Schwarz Ralph T. 
Published: March 2007

ABSTRACT

Glycosylphosphatidylinositols (GPIs) are involved in the pathogenicity of protozoan parasites and are known to induce inflammatory cytokines. However, we have previously shown that the family of six GPIs of
Toxoplasma gondii
extracted together from tachyzoites could not induce tumor necrosis factor alpha (TNF-α) secretion by macrophages, whereas GPIs individually separated from this extract by thin-layer chromatography (TLC) were able to stimulate the cells. In the present study we show that the TLC step makes it possible to eliminate inhibitors extracted together with the
T. gondii
GPIs. Among the non-GPI molecules we have isolated fatty acids able to inhibit the secretion of TNF-α induced by the
T. gondii
GPIs. Myristic and palmitic acids reduce the production of TNF-α through the inhibition of tyrosine phosphorylation of cytoplasmic proteins and the inhibition of NF-κB activation in a peroxisome proliferator-activated receptor-independent pathway and after a rapid entry into the cytoplasm of macrophages. GPIs are considered toxins inducing irreversible damage in the host, and fatty acids produced in parallel by the parasite could reduce the immune response, thus favoring the persistence of parasite infection.
Control of Quorum Sensing by a
Burkholderia pseudomallei
Multidrug Efflux Pump
Authors: Chan Ying Ying , Bian Hao Sheng , Tan Theresa May Chin , Mattmann Margrith E. , Geske Grant D. , Igarashi Jun , Hatano Tomomi , Suga Hiroaki , Blackwell Helen E. , Chua Kim Lee 
Published: March 2007

ABSTRACT

The
Burkholderia pseudomallei
KHW quorum-sensing systems produced
N
-octanoyl-homoserine lactone,
N
-decanoyl-homoserine lactone,
N
-(3-hydroxy)-octanoyl-homoserine lactone,
N
-(3-hydroxy)-decanoyl-homoserine lactone,
N
-(3-oxo)-decanoyl-homoserine lactone, and
N
-(3-oxo)-tetradecanoyl-homoserine lactone. The extracellular secretion of these acyl-homoserine lactones is dependent absolutely on the function of the
B. pseudomallei
BpeAB-OprB efflux pump.
Mycobacterial Bacilli Are Metabolically Active during Chronic Tuberculosis in Murine Lungs: Insights from Genome-Wide Transcriptional Profiling
Authors: Talaat Adel M. , Ward Sarah K. , Wu Chia-Wei , Rondon Elizabeth , Tavano Christine , Bannantine John P. , Lyons Rick , Johnston Stephen A. 
Published: March 2007

ABSTRACT

Chronic tuberculosis represents a major health problem for one-third of the world's population today. A key question relevant to chronic tuberculosis is the physiological status of
Mycobacterium tuberculosis
during this important stage of infection. To examine the molecular bases of chronic tuberculosis and the role of host immunity in mycobacterial growth, we determined the mycobacterial transcriptional profiles during chronic and reactivation phases of murine tuberculosis using in vivo microarray analysis (IVMA). Following 28 days of aerosol infection, mycobacterial counts remained stable, although the bacilli were metabolically active with a 50% active transcriptome. The expression of genes involved in lipid and carbohydrate pathways was significantly enriched during the middle stage of chronic tuberculosis, suggesting a nutrient-rich microenvironment. A total of 137 genes were significantly regulated in mid-chronic tuberculosis (45 and 60 days) compared to an early stage (14 days) of infection. Additional sets of genes, including the virulence regulator
virS
, were up-regulated during the reactivation stage, indicating their possible roles in mycobacterial resurgence. Interestingly, a set of potential transcriptional regulators was significantly induced at the late stage of chronic tuberculosis. Bioinformatic analysis identified a large number of genes that could be regulated by one of the potential transcriptional regulators encoded by rv0348, including the
sigF
operon. Taken together, IVMA provided a better definition of the transcriptional machinery activated during chronic and reactivation stages of tuberculosis and identified a novel transcriptional regulator. A similar approach can be adopted to study key stages of intracellular pathogens.
Iron-Dependent RNA-Binding Activity of
Mycobacterium tuberculosis
Aconitase
Authors: Banerjee Sharmistha , Nandyala Ashok Kumar , Raviprasad Podili , Ahmed Niyaz , Hasnain Seyed E. 
Published: March 2007

ABSTRACT

Cellular iron levels are closely monitored by iron regulatory and sensor proteins of
Mycobacterium tuberculosis
for survival inside macrophages. One such class of proteins systematically studied in eukaryotes and reported in a few prokaryotes are the iron-responsive proteins (IRPs). These IRPs bind to iron-responsive elements (IREs) present at untranslated regions (UTRs) of mRNAs and are responsible for posttranscriptional regulation of the expression of proteins involved in iron homeostasis. Amino acid sequence analysis of
M. tuberculosis
aconitase (Acn), a tricarboxylic acid (TCA) cycle enzyme, showed the presence of the conserved residues of the IRP class of proteins. We demonstrate that
M. tuberculosis
Acn is bifunctional. It is a monomeric protein that is enzymatically active in converting isocitrate to
cis
-aconitate at a broad pH range of 7 to 10 (optimum, pH 8). As evident from gel retardation assays,
M. tuberculosis
Acn also behaves like an IRP by binding to known mammalian IRE-like sequences and to predicted IRE-like sequences present at the 3′ UTR of thioredoxin (
trxC
) and the 5′ UTR of the iron-dependent repressor and activator (
ideR
) of
M. tuberculosis. M. tuberculosis
Acn when reactivated with Fe
2+
functions as a TCA cycle enzyme, but upon iron depletion by a specific iron chelator, it behaves like an IRP, binding to the selected IREs in vitro. Since iron is required for the Acn activity and inhibits the RNA-binding activity of Acn, the two activities of
M. tuberculosis
Acn are mutually exclusive. Our results demonstrate the bifunctional nature of
M. tuberculosis
Acn, pointing to its likely role in iron homeostasis.
Anaerobic Gene Expression in
Staphylococcus aureus
Authors: Fuchs Stephan , Pané-Farré Jan , Kohler Christian , Hecker Michael , Engelmann Susanne 
Published: March 2007

ABSTRACT

An investigation of gene expression in
Staphylococcus aureus
after a switch from aerobic to anaerobic growth was initiated by using the proteomic and transcriptomic approaches. In the absence of external electron acceptors like oxygen or nitrate, an induction of glycolytic enzymes was observed. At the same time the amount of tricarboxylic acid cycle enzymes was very low. NAD is regenerated by mixed acid and butanediol fermentation, as indicated by an elevated synthesis level of fermentation enzymes like lactate dehydrogenases (Ldh1 and Ldh2), alcohol dehydrogenases (AdhE and Adh), α-acetolactate decarboxylase (BudA1), acetolactate synthase (BudB), and acetoin reductase (SACOL0111) as well as an accumulation of fermentation products as lactate and acetate. Moreover, the transcription of genes possibly involved in secretion of lactate (SACOL2363) and formate (SACOL0301) was found to be induced. The formation of acetyl-coenzyme A or acetyl-phosphate might be catalyzed by pyruvate formate lyase, whose synthesis was found to be strongly induced as well. Although nitrate was not present, the expression of genes related to nitrate respiration (NarH, NarI, and NarJ) and nitrate reduction (NirD) was found to be upregulated. Of particular interest, oxygen concentration might affect the virulence properties of
S. aureus
by regulating the expression of some virulence-associated genes such as
pls
,
hly
,
splC
and
splD
,
epiG
, and
isaB
. To date, the mechanism of anaerobic gene expression in
S. aureus
has not been fully characterized. In addition to
srrA
the mRNA levels of several other regulatory genes with yet unknown functions (e.g., SACOL0201, SACOL2360, and SACOL2658) were found to be upregulated during anaerobic growth, indicating a role in the regulation of anaerobic gene expression.
Biochemical Characterization and Physiological Properties ofEscherichia coliUDP-N-Acetylmuramate:l-Alanyl-γ-d-Glutamyl-meso- Diaminopimelate Ligase
Authors: Hervé Mireille , Boniface Audrey , Gobec Stanislav , Blanot Didier , Mengin-Lecreulx Dominique 
Published: March 2007

ABSTRACTThe UDP-N-acetylmuramate:l-alanyl-γ-d-glutamyl-meso-diaminopimelate ligase (murein peptide ligase [Mpl]) is known to be a recycling enzyme allowing reincorporation into peptidoglycan (murein) of the tripeptidel-alanyl-γ-d-glutamyl-meso-diaminopimelate released during the maturation and constant remodeling of this bacterial cell wall polymer that occur during cell growth and division. Mpl adds this peptide to UDP-N-acetylmuramic acid, thereby providing an economical additional source of UDP-MurNAc-tripeptide available for de novo peptidoglycan biosynthesis. The Mpl enzyme fromEscherichia coliwas purified to homogeneity as a His-tagged form, and its kinetic properties and parameters were determined. Mpl was found to accept tri-, tetra-, and pentapeptides as substrates in vitro with similar efficiencies, but it accepted the dipeptidel-Ala-d-Glu andl-Ala very poorly. Replacement ofmeso-diaminopimelic acid byl-Lys resulted in a significant decrease in the catalytic efficacy. The effects of disruption of theE. coli mplgene and/or theldcAgene encoding theld-carboxypeptidase on peptidoglycan metabolism were investigated. The differences in the pools of UDP-MurNAc peptides and of free peptides between the wild-type and mutant strains demonstrated that the recycling activity of Mpl is not restricted to the tripeptide and that tetra- and pentapeptides are also directly reused by this process in vivo. The relatively broad substrate specificity of the Mpl ligase indicates that it is an interesting potential target for antibacterial compounds.
The Global Transcriptional Responses of
Bacillus anthracis
Sterne (34F
2
) and a Δ
sodA1
Mutant to Paraquat Reveal Metal Ion Homeostasis Imbalances during Endogenous Superoxide Stress
Authors: Passalacqua Karla D. , Bergman Nicholas H. , Lee Jung Yeop , Sherman David H. , Hanna Philip C. 
Published: March 2007

ABSTRACT

Microarray analyses were conducted to evaluate the paraquat-induced global transcriptional response of
Bacillus anthracis
Sterne (34F
2
) to varying levels of endogenous superoxide stress. Data revealed that the transcription of genes putatively involved in metal/ion transport, bacillibactin siderophore biosynthesis, the glyoxalase pathway, and oxidoreductase activity was perturbed most significantly. A
B. anthracis
mutant lacking the superoxide dismutase gene
sodA1
(Δ
sodA1
) had transcriptional responses to paraquat similar to, but notably larger than, those of the isogenic parental strain. A small, unique set of genes was found to be differentially expressed in the Δ
sodA1
mutant relative to the parental strain during growth in rich broth independently of induced oxidative stress. The bacillibactin siderophore biosynthetic genes were notably overexpressed in Sterne and Δ
sodA1
cells after treatment with paraquat. The bacillibactin siderophore itself was isolated from the supernatants and lysates of cells grown in iron-depleted medium and was detected at lower levels after treatment with paraquat. This suggests that, while transcriptional regulation of these genes is sensitive to changes in the redox environment, additional levels of posttranscriptional control may exist for bacillibactin biosynthesis, or the enzymatic siderophore pipeline may be compromised by intracellular superoxide stress or damage. The Δ
sodA1
mutant showed slower growth in a chelated iron-limiting medium but not in a metal-depleted medium, suggesting a connection between the intracellular redox state and iron/metal ion acquisition in
B. anthracis
. A double mutant lacking both the
sodA1
and
sodA2
genes (Δ
sodA1
Δ
sodA2
) was attenuated for growth in manganese-depleted medium, suggesting a slight level of redundancy between
sodA1
and
sodA2
, and a role for the
sod
genes in manganese homeostasis.