bluuebunny/crossref_metadata_2025_split huggingface dataset:
Synthesis and Selection of De Novo Proteins That Bind and Impede Cellular Functions of an Essential Mycobacterial Protein
Authors: Rao Alka , Ram Geeta , Saini Adesh Kumar , Vohra Reena , Kumar Krishan , Singh Yogendra , Ranganathan Anand 
Published: December 2006

ABSTRACT

Recent advances in nonrational and part-rational approaches to de novo peptide/protein design have shown increasing potential for development of novel peptides and proteins of therapeutic use. We demonstrated earlier the usefulness of one such approach recently developed by us, called “codon shuffling,” in creating stand-alone de novo protein libraries from which bioactive proteins could be isolated. Here, we report the synthesis and selection of codon-shuffled de novo proteins that bind to a selected
Mycobacterium tuberculosis
protein target, the histone-like protein HupB, believed to be essential for mycobacterial growth. Using a versatile bacterial two-hybrid system that entailed utilization of HupB and various codon-shuffled protein libraries as bait and prey, respectively, we were able to identify proteins that bound strongly to HupB. The observed interaction was also confirmed using an in vitro assay. One of the protein binders was expressed in
Mycobacterium smegmatis
and was shown to appreciably affect growth in the exponential phase, a period wherein HupB is selectively expressed. Furthermore, the transcription profile of
hupB
gene showed a significant reduction in the transcript quantity in mycobacterial strains expressing the protein binder. Electron microscopy of the affected mycobacteria elaborated on the extent of cell damage and hinted towards a cell division malfunction. It is our belief that a closer inspection of the obtained de novo proteins may bring about the generation of small-molecule analogs, peptidomimetics, or indeed the proteins themselves as realistic leads for drug candidates. Furthermore, our strategy is adaptable for large-scale targeting of the essential protein pool of
Mycobacterium tuberculosis
and other pathogens.
BcSAK1, a Stress-Activated Mitogen-Activated Protein Kinase, Is Involved in Vegetative Differentiation and Pathogenicity in
Botrytis cinerea
Authors: Segmüller Nadja , Ellendorf Ursula , Tudzynski Bettina , Tudzynski Paul 
Published: December 2006

ABSTRACT

The gene
bcsak1
, encoding a mitogen-activated protein kinase (MAPK) of
Botrytis cinerea
, was cloned and characterized. The protein has high homology to the yeast Hog1 and to corresponding MAPKs from filamentous fungi, but it shows unique functional features. The protein is phosphorylated under osmotic stress, specific fungicides, and oxidative stress mediated by H
2
O
2
and menadione. Northern blot analyses indicate that only a subset of typical oxidative stress response genes is regulated by BcSAK1. In contrast to most other fungal systems, Δbcsak1 mutants are significantly impaired in vegetative and pathogenic development: they are blocked in conidia formation, show increased sclerotial development, and are unable to penetrate unwounded plant tissue. These data indicate that in
B. cinerea
the stress-activated MAPK cascade is involved in essential differentiation programs.
Divergence of Protein Kinase A Catalytic Subunits in
Cryptococcus neoformans
and
Cryptococcus gattii
Illustrates Evolutionary Reconfiguration of a Signaling Cascade
Authors: Hicks Julie K. , Heitman Joseph 
Published: December 2006

ABSTRACT

Gene duplication and divergence via both the loss and gain of gene activities are powerful evolutionary forces underlying the origin of new biological functions. Here a comparative genetics approach was applied to examine the roles of protein kinase A (PKA) catalytic subunits in three closely related varieties or sibling species of the pathogenic fungus genus
Cryptococcus
. Previous studies revealed that two PKA catalytic subunits, Pka1 and Pka2, control virulence factor production and mating. However, only one of the two plays the predominant physiological role, and this function has been exchanged between Pka1 and Pka2 in strains of the
Cryptococcus neoformans
var.
grubii
serotype A lineage compared to divergent
C. neoformans
var.
neoformans
serotype D isolates. To understand the basis for this functional plasticity, here the activities of Pka1 and Pka2 were defined in the two varieties and the related sibling species
Cryptococcus gattii
by gene disruption and characterization, heterologous complementation, and analysis of serotype AD hybrid mutant strains. The findings provide evidence for a shared ancestral role of PKA in governing mating and virulence factor production and indicate that the exchange of catalytic subunit roles is attributable to loss of function. Our studies illustrate the plasticity of signaling networks enabling rapid rewiring during speciation of a clade of common human fungal pathogens.
Novel
phacB
-Encoded Cytochrome P450 Monooxygenase from
Aspergillus nidulans
with 3-Hydroxyphenylacetate 6-Hydroxylase and 3,4-Dihydroxyphenylacetate 6-Hydroxylase Activities
Authors: Ferrer-Sevillano Francisco , Fernández-Cañón José M. 
Published: December 2006

ABSTRACT

Aspergillus nidulans
catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted
phacB
, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted
phacB
strain (
ΔphacB
) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several
A. nidulans
mutant strains confirmed that the
phacB
-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The
phacB
gene is the first member of a new CYP450 subfamily (CYP504B).
The
pio
Operon Is Essential for Phototrophic Fe(II) Oxidation in
Rhodopseudomonas palustris
TIE-1
Authors: Jiao Yongqin , Newman Dianne K. 
Published: December 2006

ABSTRACT

Phototrophic Fe(II)-oxidizing bacteria couple the oxidation of ferrous iron [Fe(II)] to reductive CO
2
fixation by using light energy, but until recently, little has been understood about the molecular basis for this process. Here we report the discovery, with
Rhodopseudomonas palustris
TIE-1 as a model organism, of a three-gene operon, designated the
pio
operon (for
p
hototrophic
i
ron
o
xidation), that is necessary for phototrophic Fe(II) oxidation. The first gene in the operon,
pioA
, encodes a
c
-type cytochrome that is upregulated under Fe(II)-grown conditions. PioA contains a signal sequence and shares homology with MtrA, a decaheme
c
-type cytochrome from
Shewanella oneidensis
MR-1. The second gene,
pioB
, encodes a putative outer membrane beta-barrel protein. PioB is a homologue of MtrB from
S. oneidensis
MR-1. The third gene,
pioC
, encodes a putative high potential iron sulfur protein (HiPIP) with a twin-arginine translocation (Tat) signal sequence and is similar to the putative Fe(II) oxidoreductase (Iro) from
Acidithiobacillus ferrooxidans
. Like PioA, PioB and PioC appear to be secreted proteins. Deletion of the
pio
operon results in loss of Fe(II) oxidation activity and growth on Fe(II). Complementation studies confirm that the phenotype of this mutant is due to loss of the
pio
genes. Deletion of
pioA
alone results in loss of almost all Fe(II) oxidation activity; however, deletion of either
pioB
or
pioC
alone results in only partial loss of Fe(II) oxidation activity. Together, these results suggest that proteins encoded by the
pio
operon are essential and specific for phototrophic Fe(II) oxidation in
R. palustris
TIE-1.
Glucosylglycerate Biosynthesis in the Deepest Lineage of theBacteria: Characterization of the Thermophilic Proteins GpgS and GpgP fromPersephonella marina
Authors: Costa Joana , Empadinhas Nuno , da Costa Milton S. 
Published: December 2006

ABSTRACTThe pathway for the synthesis of glucosylglycerate (GG) in the thermophilic bacteriumPersephonella marinais proposed based on the activities of recombinant glucosyl-3-phosphoglycerate (GPG) synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP). The sequences ofgpgSandgpgPfrom the cold-adapted bacteriumMethanococcoides burtoniiwere used to identify the homologues in the genome ofP. marina, which were separately cloned and overexpressed as His-tagged proteins inEscherichia coli. The recombinant GpgS protein ofP. marina, unlike the homologue fromM. burtonii, which was specific for GDP-glucose, catalyzed the synthesis of GPG from UDP-glucose, GDP-glucose, ADP-glucose, and TDP-glucose (in order of decreasing efficiency) and fromd-3-phosphoglycerate, with maximal activity at 90°C. The recombinant GpgP protein, like theM. burtoniihomologue, dephosphorylated GPG and mannosyl-3-phosphoglycerate (MPG) to GG and mannosylglycerate, respectively, yet at high temperatures the hydrolysis of GPG was more efficient than that of MPG. Gel filtration indicates that GpgS is a dimeric protein, while GpgP is monomeric. This is the first characterization of genes and enzymes for the synthesis of GG in a thermophile.
Borrelia burgdorferi
BBA74, a Periplasmic Protein Associated with the Outer Membrane, Lacks Porin-Like Properties
Authors: Mulay Vishwaroop , Caimano Melissa J. , Liveris Dionysios , Desrosiers Daniel C. , Radolf Justin D. , Schwartz Ira 
Published: December 2006

ABSTRACT

The outer membrane of
Borrelia burgdorferi
, the causative agent of Lyme disease, contains very few integral membrane proteins, in contrast to other gram-negative bacteria. BBA74, a
Borrelia burgdorferi
plasmid-encoded protein, was proposed to be an integral outer membrane protein with putative porin function and designated as a 28-kDa outer membrane-spanning porin (Oms28). In this study, the biophysical properties of BBA74 and its subcellular localization were investigated. BBA74 is posttranslationally modified by signal peptidase I cleavage to a mature 25-kDa protein. The secondary structure of BBA74 as determined by circular dichroism spectroscopy consists of at least 78% α-helix with little β-sheet structure. BBA74 in intact
B. burgdorferi
cells was insensitive to proteinase K digestion, and indirect immunofluorescence microscopy showed that BBA74 was not exposed on the cell surface. Triton X-114 extraction of outer membrane vesicle preparations indicated that BBA74 is not an integral membrane protein. Taken together, the data indicate that BBA74 is a periplasmic, outer membrane-associated protein that lacks properties typically associated with porins.
The Alternative Sigma Factor σHIs Required for Toxin Gene Expression byBacillus anthracis
Authors: Hadjifrangiskou Maria , Chen Yahua , Koehler Theresa M. 
Published: December 2006

ABSTRACTExpression of the structural genes for the anthrax toxin proteins is coordinately controlled by host-related signals, such as elevated CO2, and thetrans-acting positive regulator AtxA. In addition to these requirements, toxin gene expression is under growth phase regulation. The transition state regulator AbrB repressesatxAexpression to influence toxin synthesis. During the late exponential phase of growth, when AbrB levels begin to decrease, toxin synthesis increases. Here we report that toxin gene expression also requires the presence ofsigH, a gene encoding the RNA polymerase sigma factor associated with development inBacillus subtilis. In the well-studiedB. subtilissystem, σHis required for sporulation and other post-exponential-phase processes and is part of a feedback control pathway forabrBexpression. Our data indicate that aBacillus anthracis sigH-null mutant is asporogenous and toxin deficient. Yet the sigma factor is required for toxin gene expression in a manner that is independent of the pathway leading to post-exponential-phase gene expression. σHpositively controlsatxAin an AbrB-independent manner. These findings, combined with previous observations, suggest that the steady-state level ofatxAexpression is critical for optimal toxin gene transcription. We propose a model whereby, under toxin-inducing growth conditions, control of toxin gene expression is fine-tuned by the independent effects of σHand AbrB on the expression ofatxA.
Nitric Oxide in Chemostat-Cultured
Escherichia coli
Is Sensed by Fnr and Other Global Regulators: Unaltered Methionine Biosynthesis Indicates Lack of S Nitrosation
Authors: Pullan Steven T. , Gidley Mark D. , Jones Richard A. , Barrett Jason , Stevanin Tania M. , Read Robert C. , Green Jeffrey , Poole Robert K. 
Published: December 2006

ABSTRACT

We previously elucidated the global transcriptional responses of
Escherichia coli
to the nitrosating agent
S
-nitrosoglutathione (GSNO) in both aerobic and anaerobic chemostats, demonstrated the expression of nitric oxide (NO)-protective mechanisms, and obtained evidence of critical thiol nitrosation. The present study was the first to examine the transcriptome of NO-exposed
E. coli
in a chemostat. Using identical conditions, we compared the GSNO stimulon with the stimulon of NO released from two NO donor compounds {3-[2-hydroxy-1-(1-methyl-ethyl)-2-nitrosohydrazino]-1-propanamine (NOC-5) and 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-
N
-methyl-1-propanamine (NOC-7)} simultaneously and demonstrated that there were marked differences in the transcriptional responses to these distinct nitrosative stresses. Exposure to NO did not induce
met
genes, suggesting that, unlike GSNO, NO does not elicit homocysteine S nitrosation and compensatory increases in methionine biosynthesis. After entry into cells, exogenous methionine provided protection from GSNO-mediated killing but not from NO-mediated killing. Anaerobic exposure to NO led to up-regulation of multiple Fnr-repressed genes and down-regulation of Fnr-activated genes, including
nrfA
, which encodes cytochrome
c
nitrite reductase, providing strong evidence that there is NO inactivation of Fnr. Other global regulators apparently affected by NO were IscR, Fur, SoxR, NsrR, and NorR. We tried to identify components of the NorR regulon by performing a microarray comparison of NO-exposed wild-type and
norR
mutant strains; only
norVW
, encoding the NO-detoxifying flavorubredoxin and its cognate reductase, were unambiguously identified. Mutation of
norV
or
norR
had no effect on
E. coli
survival in mouse macrophages. Thus, GSNO (a nitrosating agent) and NO have distinct cellular effects; NO more effectively interacts with global regulators that mediate adaptive responses to nitrosative stress but does not affect methionine requirements arising from homocysteine nitrosation.
Characterization of ResDE-DependentfnrTranscription inBacillus subtilis
Authors: Geng Hao , Zhu Yi , Mullen Karl , Zuber Cole S. , Nakano Michiko M. 
Published: December 2006

ABSTRACTThe ResD-ResE signal transduction system is required for transcription of genes involved in aerobic and anaerobic respiration inBacillus subtilis. Phosphorylated ResD (ResD∼P) interacts with target DNA to activate transcription. A strong sequence similarity was detected in promoter regions of some ResD-controlled genes includingfnrandresA. Single-base substitutions in thefnrandresApromoters were performed to determine a ResD-binding sequence. DNase I footprinting analysis indicated that ResD∼P itself does not bind tofnr, but interaction of ResD∼P with the C-terminal domain of the α subunit (αCTD) of RNA polymerase (RNAP) facilitates cooperative binding of ResD∼P and RNAP, thereby increasingfnrtranscription initiation. Consistent with this result, amino acid substitutions in αCTD, such as Y263A, K267A, A269I, or N290A, sharply reducedfnrtranscription in vivo, and the K267A αCTD protein, unlike the wild-type protein, did not increase ResD∼P binding to thefnrpromoter. Amino acid residues of αCTD required for ResD-dependentfnrtranscription, with the exception of N290, which may interact with DNA, constitute a distinct surface, suggesting that these residues likely interact with ResD∼P.
Characterization of a Novel
d
-Lyxose Isomerase from
Cohnella laevoribosii
RI-39 sp. nov
Authors: Cho Eun-Ah , Lee Dong-Woo , Cha Yun-Hwan , Lee Sang-Jae , Jung Heung-Chae , Pan Jae-Gu , Pyun Yu-Ryang 
Published: December 2006

ABSTRACT

A newly isolated bacterium,
Cohnella laevoribosii
RI-39, could grow in a defined medium with
l
-ribose as the sole carbon source. A 21-kDa protein isomerizing
l
-ribose to
l
-ribulose, as well as
d
-lyxose to
d
-xylulose, was purified to homogeneity from this bacterium. Based on the N-terminal and internal amino acid sequences of the purified enzyme obtained by N-terminal sequencing and quantitative time of flight mass spectrometry-mass spectrometry analyses, a 549-bp gene (
lyxA
) encoding
d
-lyxose (
l
-ribose) isomerase was cloned and expressed in
Escherichia coli
. The purified endogenous enzyme and the recombinant enzyme formed homodimers that were activated by Mn
2+
.
C. laevoribosii
d
-lyxose (
l
-ribose) isomerase (CLLI) exhibits maximal activity at pH 6.5 and 70°C in the presence of Mn
2+
for
d
-lyxose and
l
-ribose, and its isoelectric point (pI) is 4.2 (calculated pI, 4.9). The enzyme is specific for
d
-lyxose,
l
-ribose, and
d
-mannose, with apparent

K
m

values of 22.4 ± 1.5 mM, 121.7 ± 10.8 mM, and 34.0 ± 1.1 mM, respectively. The catalytic efficiencies (
k
cat
/

K
m

) of CLLI were 84.9 ± 5.8 mM
−1
s
−1
for
d
-lyxose (
V
max
, 5,434.8 U mg
−1
), 0.2 mM
−1
s
−1
for
l
-ribose (
V
max
, 75.5 ± 6.0 U mg
−1
), and 1.4 ± 0.1 mM
−1
s
−1
for
d
-mannose (
V
max
, 131.8 ± 7.4 U mg
−1
). The ability of
lyxA
to permit
E. coli
cells to grow on
d
-lyxose and
l
-ribose and homology searches of other sugar-related enzymes, as well as previously described sugar isomerases, suggest that CLLI is a novel type of rare sugar isomerase.
Mutations of the Act Promoter in
Myxococcus xanthus
Authors: Gronewold Thomas M. A. , Kaiser Dale 
Published: December 2006

ABSTRACT

Mutations within the −12 and −24 elements provide evidence that the
act
promoter is recognized by sigma-54 RNA polymerase. Deletion of the −20 base pair, which lies between the two conserved elements of sigma-54 promoters, decreased expression by 90%. In addition, mutation of a potential enhancer sequence, around −120, led to an 80% reduction in
act
gene expression.
actB
, the second gene in the
act
operon, encodes a sigma-54 activator protein that is proposed to be an enhancer-binding protein for the
act
operon. All
act
genes,
actA
to
actE
, are expressed together and constitute an operon, because an in-frame deletion of
actB
decreased expression of
actA
and
actE
to the same extent. After an initially slow phase of
act
operon expression, which depends on FruA, there is a rapid phase. The rapid phase is shown to be due to the activation of the operon expression by ActB, which completes a positive feedback loop. That loop appears to be nested within a larger positive loop in which ActB is activated by the C signal via ActA, and the
act
operon activates transcription of the
csgA
gene. We propose that, as cells engage in more C signaling, positive feedback raises the number of C-signal molecules per cell and drives the process of fruiting body development forward.
YjjG, a dUMP Phosphatase, Is Critical for Thymine Utilization by
Escherichia coli
K-12
Authors: Weiss Bernard 
Published: December 2006

ABSTRACT

Exogenous thymine must be converted to thymidine to enable a
thyA
(thymidylate synthase) mutant to grow. The deoxyribose in the thymidine comes from dUMP, which must first be dephosphorylated. The nucleotidase YjjG is critical for this step. A
yjjG thyA
mutant cannot use thymine for growth on a glucose minimal medium.
Growth Phase Regulation of
Vibrio cholerae
RTX Toxin Export
Authors: Boardman Bethany Kay , Meehan Brian M. , Fullner Satchell Karla J. 
Published: December 2006

ABSTRACT

Vibrio cholerae
, the causative agent of the severe diarrheal disease cholera, secretes several “accessory” toxins, including RTX toxin, which causes the cross-linking of the actin cytoskeleton. RTX toxin is exported to the extracellular milieu by an atypical type I secretion system (T1SS), and we previously noted that RTX-associated activity is detectable only in supernatant fluids from log phase cultures. Here, we investigate the mechanisms for regulating RTX toxin activity in supernatant fluids. We find that exported proteases are capable of destroying RTX activity and may therefore play a role in the growth phase regulation of toxin activity. We determined that the absence of RTX toxin in stationary-phase culture supernatant fluids is also due to a lack of toxin secretion and not attributable to solely proteolytic degradation. We ascertained that the T1SS apparatus is regulated at the transcriptional level by growth phase control that is independent of quorum sensing, unlike other virulence factors of
V. cholerae
. Additionally, in stationary-phase cultures, all RTX toxin activity is associated with bacterial membranes or outer membrane vesicles.
Nonsynonymous Coding Single-Nucleotide Polymorphisms Spanning the Genome in Relation to Glioblastoma Survival and Age at Diagnosis
Authors: Wrensch Margaret , McMillan Alex , Wiencke John , Wiemels Joe , Kelsey Karl , Patoka Joe , Jones Hywel , Carlton Victoria , Miike Rei , Sison Jennette , Moghadassi Michelle , Prados Michael 
Published: January 2007

AbstractPurpose: Our aim was to discover possible inherited factors associated with glioblastoma age at diagnosis and survival. Although new genotyping technologies allow greatly expanded exploration of such factors, they pose many challenges.Experimental Design: In this pilot study, we (a) genotyped 112 newly diagnosed glioblastoma patients ascertained through a population-based study (group 1) with the ParAllele assay panel of ∼10,000 nonsynonymous coding single-nucleotide polymorphisms (SNP), (b) used several statistical and bioinformatic techniques to identify 17 SNPs potentially related to either glioblastoma age at diagnosis or survival, and (c) genotyped 16 of these SNPs using conventional PCR methods in an independent group of 195 glioblastoma patients (group 2).Results: In group 2, only one of the 16 SNPs, rs8057643 (located on 16p13.2), was significantly associated with glioblastoma age at diagnosis (nominal P = 0.0017; Bonferroni corrected P = 0.054). Median ages at diagnosis for those with 0, 1, or 2 T alleles were 66, 57, and 59 years in group 1 and 64, 57, and 55 years in group 2 (combined P = 0.001). Furthermore, Cox regression analyses of time to death with number of T alleles adjusted for gender and patient group yielded a hazard ratio of 0.82 (95% confidence interval, 0.68-0.98; P = 0.03).Conclusions: Although limited by a relatively small sample size, this pilot study, using well-characterized, unambiguous disease characteristics, illustrates the necessity of independent replication owing to the likelihood of false positives. Several other challenges are discussed, including attempts to incorporate information on the potential functional importance of SNPs in genome-disease association studies.