bluuebunny/crossref_metadata_2025_split huggingface dataset:
Relevant Double Mutations in Bioengineered
Streptomyces clavuligerus
Deacetoxycephalosporin C Synthase Result in Higher Binding Specificities Which Improve Penicillin Bioconversion
Authors: Goo Kian Sim , Chua Chun Song , Sim Tiow-Suan 
Published: December 2007

ABSTRACT

Streptomyces clavuligerus
deacetoxycephalosporin C synthase (ScDAOCS) is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid, which is a precursor for cephalosporin synthesis. Single mutations of six amino acid residues, V275, C281, N304, I305, R306, and R307, were previously shown to result in enhanced levels of ampicillin conversion, with activities ranging from 129 to 346% of the wild-type activity. In this study, these mutations were paired to investigate their effects on enzyme catalysis. The bioassay results showed that the C-terminal mutations (N304X [where X is alanine, leucine, methionine, lysine, or arginine], I305M, R306L, and R307L) in combination with C281Y substantially increased the conversion of ampicillin; the activity was up to 491% of the wild-type activity. Similar improvements were observed for converting carbenicillin (up to 1,347% of the wild-type activity) and phenethicillin (up to 1,109% of the wild-type activity). Interestingly, the N304X R306L double mutants exhibited lower activities for penicillin G conversion, and activities that were 40 to 114% of wild-type enzyme activity were detected. Based on kinetic studies using ampicillin, it was clear that the increases in the activities of the double mutants relative to those of the corresponding single mutants were due to enhanced substrate binding affinities. These results also validated the finding that the N304R and I305M mutations are ideal for increasing the substrate binding affinity and turnover rate of the enzyme, respectively. This study provided further insight into the structure-function interaction of ScDAOCS with different penicillin substrates, thus providing a useful platform for further rational modification of its enzymatic properties.
Sensitivity of an
Acinetobacter baylyi mpl
Mutant to DNA Damage
Authors: Chakravorty Adityarup , Klovstad Martha , Peterson Greg , Lindeman Robin E. , Gregg-Jolly Leslie A. 
Published: December 2007

ABSTRACT

A mutant derived from
Acinetobacter baylyi
ADP1 was isolated from a screen for genes involved in the response to DNA damage. This derivative has an insertion in the
mpl
gene which encodes a peptidoglycan-recycling protein. We demonstrate that the insertion renders cells sensitive to mitomycin C and to UV.
CTA4
Transcription Factor Mediates Induction of Nitrosative Stress Response in
Candida albicans
Authors: Chiranand Wiriya , McLeod Ian , Zhou Huaijin , Lynn Jed J. , Vega Luis A. , Myers Hadley , Yates John R. , Lorenz Michael C. , Gustin Michael C. 
Published: December 2007

ABSTRACT

This work has identified regulatory elements in the major fungal pathogen
Candida albicans
that enable response to nitrosative stress. Nitric oxide (NO) is generated by macrophages of the host immune system and commensal bacteria, and the ability to resist its toxicity is one adaptation that promotes survival of
C. albicans
inside the human body. Exposing
C. albicans
to NO induces upregulation of the flavohemoglobin Yhb1p. This protein confers protection by enzymatically converting NO to harmless nitrate, but it is unknown how
C. albicans
is able to detect NO in its environment and thus initiate this defense only as needed. We analyzed this problem by incrementally mutating the
YHB1
regulatory region to identify a nitric oxide-responsive element (NORE) that is required for NO sensitivity. Five transcription factor candidates of the Zn(II)2-Cys6 family were then isolated from crude whole-cell extracts by using magnetic beads coated with this DNA element. Of the five, only deletion of the
CTA4
gene prevented induction of
YHB1
transcription during nitrosative stress and caused growth sensitivity to the NO donor dipropylenetriamine NONOate; Cta4p associates in vivo with NORE DNA from the
YHB1
regulatory region. Deletion of
CTA4
caused a small but significant decrease in virulence. A
CTA4
-dependent putative sulfite transporter encoded by
SSU1
is also implicated in NO response, but
C. albicans ssu1
mutants were not sensitive to NO, in contrast to findings in
Saccharomyces cerevisiae
. Cta4p is the first protein found to be necessary for initiating NO response in
C. albicans
.
Disruption of the PfPK7Gene Impairs Schizogony and Sporogony in the Human Malaria ParasitePlasmodium falciparum
Authors: Dorin-Semblat Dominique , Sicard Audrey , Doerig Caroline , Ranford-Cartwright Lisa , Doerig Christian 
Published: December 2007

ABSTRACTPfPK7 is an orphan protein kinase ofPlasmodium falciparumwith maximal homology to MEK3/6 and to fungal protein kinase A proteins in its C-terminal and N-terminal regions, respectively. We showed previously that recombinant PfPK7 is active on various substrates but is unable to phosphorylate thePlasmodium falciparummitogen-activated protein kinase homologues, suggesting that it is not a MEK functional homologue. Using a reverse genetics approach to investigate the function of this enzyme in live parasites, we now show that PfPK7−parasite clones display phenotypes at two stages of their life cycle: first, a decrease in the rate of asexual growth in erythrocytes associated with a lower number of daughter merozoites generated per schizont, and second, a dramatic reduction in the ability to produce oocysts in the mosquito vector. A normal asexual growth rate and the ability to produce oocysts are restored if a functional copy of the PfPK7gene is reintroduced into the PfPK7−parasites. Hence, PfPK7 is involved in a pathway that regulates parasite proliferation and development.
The L-Type Calcium Ion Channel Cch1 Affects Ascospore Discharge and Mycelial Growth in the Filamentous FungusGibberella zeae(AnamorphFusarium graminearum)
Authors: Hallen Heather E. , Trail Frances 
Published: December 2007

ABSTRACTCch1, a putative voltage-gated calcium ion channel, was investigated for its role in ascus development inGibberella zeae. Gene replacement mutants ofCCH1were generated and found to have asci which did not forcibly discharge spores, although morphologically ascus and ascospore development in the majority of asci appeared normal. Additionally, mycelial growth was significantly slower, and sexual development was slightly delayed in the mutant; mutant mycelia showed a distinctive fluffy morphology, and no cirrhi were produced. Wheat infected with Δcch1mutants developed symptoms comparable to wheat infected with the wild type; however, the mutants showed a reduced ability to protect the infected stalk from colonization by saprobic fungi. Transcriptional analysis of gene expression in mutants using the AffymetrixFusariummicroarray showed 2,449 genes with significant, twofold or greater, changes in transcript abundance across a developmental series. This work extends the role ofCCH1to forcible spore discharge inG. zeaeand suggests that this channel has subtle effects on growth and development.
Excystation of
Eimeria tenella
Sporozoites Impaired by Antibody Recognizing Gametocyte/Oocyst Antigens GAM22 and GAM56
Authors: Krücken Jürgen , Hosse Ralf J. , Mouafo Aimdip N. , Entzeroth Rolf , Bierbaum Stefan , Marinovski Predrag , Hain Karolina , Greif Gisela , Wunderlich Frank 
Published: December 2007

ABSTRACT

Eimeria tenella
is the causative agent of coccidiosis in poultry. Infection of the chicken intestine begins with ingestion of sporulated oocysts releasing sporocysts, which in turn release invasive sporozoites. The monoclonal antibody E2E5 recognizes wall-forming body type II (WFBII) in gametocytes and the WFBII-derived inner wall of oocysts. Here we describe that this antibody also binds to the stieda body of sporocysts and significantly impairs in vitro excystation of sporozoites. Using affinity chromatography and protein sequence analysis, E2E5 is shown to recognize EtGAM56, the
E. tenella
ortholog of the
Eimeria maxima
gametocyte-specific GAM56 protein. In addition, this antibody was used to screen a genomic phage display library presenting
E. tenella
antigens as fusion proteins with the gene VIII product on the surfaces of phagemid particles and identified the novel 22-kDa histidine- and proline-rich protein EtGAM22. The Et
gam22
mRNA is expressed predominantly at the gametocyte stage, as detected by Northern blotting. Southern blot analysis in combination with data from the
E. tenella
genome project revealed that Et
gam22
is an intronless multicopy gene, with approximately 12 to 22 copies in head-to-tail arrangement. Conspicuously, Et
gam56
is also intronless and is localized adjacent to another
gam56
-like gene, Et
gam59
. Our data suggest that amplification is common for genes encoding oocyst wall proteins.
Candida albicans
Als Adhesins Have Conserved Amyloid-Forming Sequences
Authors: Otoo Henry N. , Lee Kyeng Gea , Qiu Weigang , Lipke Peter N. 
Published: December 2007

ABSTRACT

The cell wall-bound Als adhesins of
Candida albicans
mediate both yeast-to-host tissue adherence and yeast aggregation. This aggregation is amyloid-like, with self-propagating secondary-structure changes, amyloid-characteristic dye binding, and induced birefringence (J. M. Rauceo, N. K. Gaur, K. G. Lee, J. E. Edwards, S. A. Klotz, and P. N. Lipke, Infect. Immun. 72:4948-4955, 2004). Therefore, we determined whether Als proteins could form amyloid fibers with properties like those in cellular aggregation. The β-aggregation predictor TANGO identified a heptapeptide sequence present in a highly conserved sequence with amyloid-forming potential in Als1p, Als3p, and Als5p. A tridecapeptide containing this sequence formed fibers that bound Congo red and thioflavin T and had characteristic amyloid morphology. Als5p
20-431
and Als5p
20-664
, large fragments of Als5p containing the amyloid sequence, also formed amyloid-like fibers and bound Congo red under native conditions.

K
a

/

K
s

analysis showed that the amyloid-forming sequences are highly conserved in Als proteins and evolve more slowly than other regions of the proteins. Therefore, amyloid-forming ability itself is conserved in these proteins.
Atf2 Transcription Factor Binds to the APP1 Promoter in
Cryptococcus neoformans
: Stimulatory Effect of Diacylglycerol
Authors: Tommasino Nicola , Villani Maristella , Qureshi Asfia , Henry Jennifer , Luberto Chiara , Del Poeta Maurizio 
Published: December 2007

ABSTRACT

The fungus
Cryptococcus neoformans
is an environmental human pathogen which enters the lung via the respiratory tract and produces a unique protein, called antiphagocytic protein 1 (App1), that protects it from phagocytosis by macrophages. In previous studies, we proposed genetic evidences that transcription of
APP1
is controlled by the enzymatic reaction catalyzed by inositol phosphorylceramide synthase 1 (Ipc1) via the production of diacylglycerol through the activating transcription factor 2 (Atf2). We investigated here the mechanism by which Atf2 binds to the
APP1
promoter in vitro and in vivo. To this end, we produced Atf2 recombinant proteins (rAtf2) and found that rAtf2 binds to ATF
cis
-acting element present in the
APP1
promoter. Indeed, mutation of two key nucleotides in the ATF consensus sequence abolishes the binding of rAtf2 to the
APP1
promoter. Next, we produced
C. neoformans
strains with a hemagglutinin-tagged
ATF2
gene and showed that endogenous Atf2 binds to
APP1
promoter in vivo. Finally, by a novel DNA protein-binding precipitation assay, we showed that treatment with 1,2-dioctanoylglycerol positively increases binding of Atf2-
APP1
promoter in vivo. These studies provide new insights into the molecular mechanism by which Atf2 regulates
APP1
transcription in vivo with important implications for a better understanding of how
C. neoformans
escapes the phagocytic process.
The Cytoplasmic Region of α-1,6-Mannosyltransferase Mnn9p Is Crucial for Retrograde Transport from the Golgi Apparatus to the Endoplasmic Reticulum in
Saccharomyce
s
cerevisiae
Authors: Okamoto Michiyo , Yoko-o Takehiko , Miyakawa Tokichi , Jigami Yoshifumi 
Published: December 2007

ABSTRACT

In
Saccharomyces cerevisiae
, Och1p and Mnn9p mannosyltransferases are localized in the
cis
-Golgi. Attempts to live image Och1p and Mnn9p tagged with green fluorescent protein or red fluorescent protein, respectively, using a high-performance confocal laser scanning microscope system resulted in simultaneous visualization of the native proteins in a living cell. Our observations revealed that Och1p and Mnn9p are not always colocalized to the same cisternae. The difference in the dynamics of these mannosyltransferases may reflect differences in the mechanisms for their retention in the
cis
-Golgi, since it has been reported that Mnn9p cycles between the endoplasmic reticulum and the
cis
-Golgi whereas Och1p does not (Z. Todorow, A. Spang, E. Carmack, J. Yates, and R. Schekman, Proc. Natl. Acad. Sci. USA
97:
13643-13648, 2000). We investigated the localization of chimeric proteins of Mnn9p and Och1p in
sec12
and
erd1
mutant cells. A chimeric protein, M16/O16, which consists of the N-terminal cytoplasmic region of Mnn9p and the transmembrane and luminal region of Och1p, behaved like Mnn9p, suggesting that the N-terminal cytoplasmic region is important for the intracellular dynamics of Mnn9p. This observation is supported by results from subcellular-fractionation experiments. Mutational analysis revealed that two arginine residues in the N-terminal region of Mnn9p are important for the chimeric protein to cycle between the endoplasmic reticulum and the Golgi apparatus.
Impact of Ammonium Permeases MepA, MepB, and MepC on Nitrogen-Regulated Secondary Metabolism in
Fusarium fujikuroi
Authors: Teichert Sabine , Rutherford Julian C. , Wottawa Marieke , Heitman Joseph , Tudzynski Bettina 
Published: December 2007

ABSTRACT

In
Fusarium fujikuroi
, the production of gibberellins and bikaverin is repressed by nitrogen sources such as glutamine or ammonium. Sensing and uptake of ammonium by specific permeases play key roles in nitrogen metabolism. Here, we describe the cloning of three ammonium permease genes,
mepA, mepB
, and
mepC
, and their participation in ammonium uptake and signal transduction in
F. fujikuroi
. The expression of all three genes is strictly regulated by the nitrogen regulator AreA. Severe growth defects of Δ
mepB
mutants on low-ammonium medium and methylamine uptake studies suggest that MepB functions as the main ammonium permease in
F. fujikuroi
. In Δ
mepB
mutants, nitrogen-regulated genes such as the gibberellin and bikaverin biosynthetic genes are derepressed in spite of high extracellular ammonium concentrations.
mepA mepB
and
mepC mepB
double mutants show a similar phenotype as Δ
mepB
mutants. All three
F. fujikuroi mep
genes fully complemented the
Saccharomyces cerevisiae mep1 mep2 mep3
triple mutant to restore growth on low-ammonium medium, whereas only MepA and MepC restored pseudohyphal growth in the
mep2/mep2
mutant. Overexpression of
mepC
in the Δ
mepB
mutants partially suppressed the growth defect but did not prevent derepression of AreA-regulated genes. These studies provide evidence that MepB functions as a regulatory element in a nitrogen sensing system in
F. fujikuroi
yet does not provide the sensor activity of Mep2 in yeast, indicating differences in the mechanisms by which nitrogen is sensed in
S. cerevisiae
and
F. fujikuroi
.
Interaction between Two Putative Glycosyltransferases Is Required for Glycosylation of a Serine-Rich Streptococcal Adhesin
Authors: Bu Su , Li Yirong , Zhou Meixian , Azadin Parastoo , Zeng Meiqin , Fives-Taylor Paula , Wu Hui 
Published: December 2007

ABSTRACT

Fap1, a serine-rich glycoprotein, is essential for fimbrial biogenesis and biofilm formation of
Streptococcus parasanguinis
(formerly
S. parasanguis
). Fap1-like proteins are conserved in many streptococci and staphylococci and have been implicated in bacterial virulence. Fap1 contains two serine-rich repeat regions that are modified by O-linked glycosylation. A seven-gene cluster has been identified, and this cluster is implicated in Fap1 biogenesis. In this study, we investigated the initial step of Fap1 glycosylation by using a recombinant Fap1 as a model. This recombinant molecule has the same monosaccharide composition profile as the native Fap1 protein. Glycosyl linkage analyses indicated that
N
-acetylglucosamine (GlcNAc) is among the first group of sugar residues transferred to the Fap1 peptide. Two putative glycosyltransferases, Gtf1 and Gtf2, were essential for the glycosylation of Fap1 with GlcNAc-containing oligosaccharide(s) in both
S. parasanguinis
as well as in the Fap1 glycosylation system in
Escherichia coli
. Yeast two-hybrid analysis as well as in vitro and in vivo glutathione
S
-transferase pull-down assays demonstrated the two putative glycosyltransferases interacted with each other. The interaction domain was mapped to an N-terminal region of Gtf1 that was required for the Fap1 glycosylation. The data in this study suggested that the formation of the Gtf1 and Gtf2 complex was required for the initiation of the Fap1 glycosylation and that the N-terminal region of Gtf1 was necessary.
Characterization of a New Rhamnogalacturonan Acetyl Esterase from
Bacillus halodurans
C-125 with a New Putative Carbohydrate Binding Domain
Authors: Navarro-Fernández José , Martínez-Martínez Irene , Montoro-García Silvia , García-Carmona Francisco , Takami Hideto , Sánchez-Ferrer Álvaro 
Published: December 2007

ABSTRACT

BH1115 is a gene from
Bacillus halodurans
strain C-125 that hypothetically encodes a rhamnogalacturonan acetyl esterase (RGAE) of the CE-12 family. As confirmation, this gene was cloned, and the product was expressed in
Escherichia coli
strain Rosetta (DE3) cells and purified. The enzyme obtained was monomeric, with a molecular mass of 45 kDa, and exhibited alkaliphilic properties. A study of the inhibition of the activity by some modulators confirmed that the catalytic triad for the esterase activity was Ser-His-Asp. This enzyme also presents broad substrate specificity and is active toward 7-aminocephalosporanic acid, cephalosporin C,
p
-nitrophenyl acetate, β-naphthyl acetate, glucose pentaacetate, and acetylated xylan. Moreover, RGAE from
B. halodurans
achieves a synergistic effect with xylanase A toward acetylated xylan. As a member of the SGNH family, it does not adopt the common α/β hydrolase fold. The homology between the folds of RGAE from
Aspergillus aculeatus
and the hypothetical YxiM precursor from
Bacillus subtilis
, which both belong to the SGNH family, illustrates the divergence of such proteins from a common ancestor. Furthermore, the enzyme possesses a putative substrate binding region at the N terminus of the protein which has never been described to date for any RGAE.
Gene Acquisition at the Insertion Site for SCC
mec
, the Genomic Island Conferring Methicillin Resistance in
Staphylococcus aureus
Authors: Noto Michael J. , Kreiswirth Barry N. , Monk Alastair B. , Archer Gordon L. 
Published: December 2007

ABSTRACT

Staphylococcus aureus
becomes resistant to methicillin by acquiring a genomic island, known as staphylococcal chromosome cassette
mec
(SCC
mec
), which contains the methicillin resistance determinant,
mecA
. SCC
mec
is site-specifically integrated into the staphylococcal chromosome at a locus known as the SCC
mec
attachment site (
attB
). In an effort to gain a better understanding of the potential that methicillin-sensitive
S. aureus
(MSSA) isolates have for acquiring SCC
mec
, the nucleotide sequences of
attB
and surrounding DNA regions were examined in a diverse collection of 42 MSSA isolates. The chromosomal region surrounding
attB
varied among the isolates studied and appears to be a common insertion point for acquired foreign DNA. Insertions of up to 15.1 kb were found containing open reading frames with homology to enterotoxin genes, restriction-modification systems, transposases, and several sequences that have not been previously described in staphylococci. Two groups, containing eight and four isolates, had sequences found in known SCC
mec
elements, suggesting SCC
mec
elements may have evolved through repeated DNA insertions at this locus. In addition, the
attB
sequences of the majority of MSSA isolates in this collection differ from the
attB
sequences of strains for which integrase-mediated SCC
mec
insertion or excision has been demonstrated, suggesting that some
S. aureus
isolates may lack the ability to site-specifically integrate SCC
me
c into their chromosomes.
Pseudomonas aeruginosaPqsA Is an Anthranilate-Coenzyme A Ligase
Authors: Coleman James P. , Hudson L. Lynn , McKnight Susan L. , Farrow John M. , Calfee M. Worth , Lindsey Claire A. , Pesci Everett C. 
Published: December 2007

ABSTRACTPseudomonas aeruginosais an opportunistic human pathogen which relies on several intercellular signaling systems for optimum population density-dependent regulation of virulence genes. ThePseudomonasquinolone signal (PQS) is a 3-hydroxy-4-quinolone with a 2-alkyl substitution which is synthesized by the condensation of anthranilic acid with a 3-keto-fatty acid. ThepqsABCDEoperon has been identified as being necessary for PQS production, and thepqsAgene encodes a predicted protein with homology to acyl coenzyme A (acyl-CoA) ligases. In order to elucidate the first step of the 4-quinolone synthesis pathway inP. aeruginosa, we have characterized the function of thepqsAgene product. Extracts prepared fromEscherichia coliexpressing PqsA were shown to catalyze the formation of anthraniloyl-CoA from anthranilate, ATP, and CoA. The PqsA protein was purified as a recombinant His-tagged polypeptide, and this protein was shown to have anthranilate-CoA ligase activity. The enzyme was active on a variety of aromatic substrates, including benzoate and chloro and fluoro derivatives of anthranilate. Inhibition of PQS formation in vivo was observed for the chloro- and fluoroanthranilate derivatives, as well as for several analogs which were not PqsA enzymatic substrates. These results indicate that the PqsA protein is responsible for priming anthranilate for entry into the PQS biosynthetic pathway and that this enzyme may serve as a useful in vitro indicator for potential agents to disrupt quinolone signaling inP. aeruginosa.
Xylanase Attachment to the Cell Wall of the Hyperthermophilic Bacterium
Thermotoga maritima
Authors: Liebl Wolfgang , Winterhalter Christoph , Baumeister Wolfgang , Armbrecht Martin , Valdez Michael 
Published: December 2007

ABSTRACT

The cellular localization and processing of the endo-xylanases (1,4-β-
d
-xylan-xylanohydrolase; EC 3.2.1.8) of the hyperthermophile
Thermotoga maritima
were investigated, in particular with respect to the unusual outer membrane (“toga”) of this gram-negative bacterium. XynB (40 kDa) was detected in the periplasmic fraction of
T. maritima
cells and in the culture supernatant. XynA (120 kDa) was partially released to the surrounding medium, but most XynA remained cell associated. Immunogold labeling of thin sections revealed that cell-bound XynA was localized mainly in the outer membranes of
T. maritima
cells. Amino-terminal sequencing of purified membrane-bound XynA revealed processing of the signal peptide after the eighth residue, thereby leaving the hydrophobic core of the signal peptide attached to the enzyme. This mode of processing is reminiscent of type IV prepilin signal peptide cleavage. Removal of the entire XynA signal peptide was necessary for release from the cell because enzyme purified from the culture supernatant lacked 44 residues at the N terminus, including the hydrophobic part of the signal peptide. We conclude that toga association of XynA is mediated by residues 9 to 44 of the signal peptide. The biochemical and electron microscopic localization studies together with the amino-terminal processing data indicate that XynA is held at the cell surface of
T. maritima
via a hydrophobic peptide anchor, which is highly unusual for an outer membrane protein.