bluuebunny/crossref_metadata_2025_split huggingface dataset:
High Glycolytic Flux Improves Pyruvate Production by a Metabolically Engineered
Escherichia coli
Strain
Authors: Zhu Yihui , Eiteman Mark A. , Altman Ronni , Altman Elliot 
Published: September 2008

ABSTRACT

We report pyruvate formation in
Escherichia coli
strain ALS929 containing mutations in the
aceEF
,
pfl
,
poxB
,
pps
, and
ldhA
genes which encode, respectively, the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, phosphoenolpyruvate synthase, and lactate dehydrogenase. The glycolytic rate and pyruvate productivity were compared using glucose-, acetate-, nitrogen-, or phosphorus-limited chemostats at a growth rate of 0.15 h
−1
. Of these four nutrient limitation conditions, growth under acetate limitation resulted in the highest glycolytic flux (1.60 g/g · h), pyruvate formation rate (1.11 g/g · h), and pyruvate yield (0.70 g/g). Additional mutations in
atpFH
and
arcA
(strain ALS1059) further elevated the steady-state glycolytic flux to 2.38 g/g · h in an acetate-limited chemostat, with heterologous NADH oxidase expression causing only modest additional improvement. A fed-batch process with strain ALS1059 using defined medium with 5 mM betaine as osmoprotectant and an exponential feeding rate of 0.15 h
−1
achieved 90 g/liter pyruvate, with an overall productivity of 2.1 g/liter · h and yield of 0.68 g/g.
Oxygen-Dependent Transcriptional Regulator Hap1p Limits Glucose Uptake by Repressing the Expression of the Major Glucose Transporter Gene
RAG1
in
Kluyveromyces lactis
Authors: Bao Wei-Guo , Guiard Bernard , Fang Zi-An , Donnini Claudia , Gervais Michel , Passos Flavia M. Lopes , Ferrero Iliana , Fukuhara Hiroshi , Bolotin-Fukuhara Monique 
Published: September 2008

ABSTRACT

The
HAP1
(
CYP1
) gene product of
Saccharomyces cerevisiae
is known to regulate the transcription of many genes in response to oxygen availability. This response varies according to yeast species, probably reflecting the specific nature of their oxidative metabolism. It is suspected that a difference in the interaction of Hap1p with its target genes may explain some of the species-related variation in oxygen responses. As opposed to the fermentative
S. cerevisiae
,
Kluyveromyces lactis
is an aerobic yeast species which shows different oxygen responses. We examined the role of the
HAP1
-equivalent gene (Kl
HAP1
) in
K. lactis
. KlHap1p showed a number of sequence features and some gene targets (such as Kl
CYC1
) in common with its
S. cerevisiae
counterpart, and Kl
HAP1
was capable of complementing the
hap1
mutation. However, the Kl
HAP1
disruptant showed temperature-sensitive growth on glucose, especially at low glucose concentrations. At normal temperature, 28°C, the mutant grew well, the colony size being even greater than that of the wild type. The most striking observation was that KlHap1p repressed the expression of the major glucose transporter gene
RAG1
and reduced the glucose uptake rate. This suggested an involvement of KlHap1p in the regulation of glycolytic flux through the glucose transport system. The ΔKl
hap1
mutant showed an increased ability to produce ethanol during aerobic growth, indicating a possible transformation of its physiological property to Crabtree positivity or partial Crabtree positivity. Dual roles of KlHap1p in activating respiration and repressing fermentation may be seen as a basis of the Crabtree-negative physiology of
K. lactis
.
ESCRT-III Protein Snf7 Mediates High-Level Expression of the
SUC2
Gene via the Rim101 Pathway
Authors: Weiß Peter , Huppert Stefanie , Kölling Ralf 
Published: September 2008

ABSTRACT

The yeast (
Saccharomyces cerevisiae
) Snf7 family consists of six highly charged, coiled-coil-forming proteins involved in multivesicular body (MVB) formation. Although all proteins perform a common function at late endosomes, individual mutants also show distinct phenotypes. This suggests that Snf7 homologues have additional functions separate from their role in MVB formation. In this report, we explored the molecular basis for the
s
ucrose-
n
on
f
ermenting phenotype of
snf7
mutants. Our Northern blotting experiments provide evidence that Snf7 is involved in the regulation of
SUC2
transcription. The Snf7-dependent regulation of
SUC2
transcription does not appear to involve the transcription factor Mig1, since Mig1 phosphorylation after glucose derepression was not affected in a Δs
nf7
mutant. Instead, we show that Snf7 influences
SUC2
expression by regulating the level of the transcription factor Nrg1. Snf7 exerts its effects on Nrg1 levels through activation of the transcription factor Rim101, which is part of the yeast alkaline response pathway (“Rim101 pathway”). This is supported by the findings that deletion of
RIM101
or overexpression of
NRG1
from the
ADH1
promoter leads to the same
SUC2
expression level as deletion of
SNF7
. In addition, deletion of other components of the Rim101 pathway, like
RIM13
and
RIM20
, led to the same growth phenotype on raffinose media as deletion of
SNF7
. Furthermore, Snf7 turned out to be dispensable for
SUC2
expression in an
NRG1
deletion background. Thus, the effects of Snf7 on
SUC2
expression can be completely accounted for by its effect on Nrg1 levels.
The Synthetic Genetic Network around
PKC1
Identifies Novel Modulators and Components of Protein Kinase C Signaling in
Saccharomyces cerevisiae
Authors: Krause Sue A. , Xu Hong , Gray Joseph V. 
Published: September 2008

ABSTRACT

Budding yeast
Saccharomyces cerevisiae
contains one protein kinase C (PKC) isozyme encoded by the essential gene
PKC1
. Pkc1 is activated by the small GTPase Rho1 and plays a central role in the cell wall integrity (CWI) signaling pathway. This pathway acts primarily to remodel the cell surface throughout the normal life cycle and upon various environmental stresses. The pathway is heavily branched, with multiple nonessential branches feeding into and out of the central essential Rho1-Pkc1 module. In an attempt to identify novel components and modifiers of CWI signaling, we determined the synthetic lethal genetic network around
PKC1
by using dominant-negative synthetic genetic array analysis. The resulting mutants are hypersensitive to lowered Pkc1 activity. The corresponding 21 nonessential genes are closely related to CWI function: 14 behave in a chemical-genetic epistasis test as acting in the pathway, and 6 of these genes encode known components. Twelve of the 21 null mutants display elevated CWI reporter activity, consistent with the idea that the pathway is activated by and compensates for loss of the gene products. Four of the 21 mutants display low CWI reporter activity, consistent with the idea that the pathway is compromised in these mutants. One of the latter group of mutants lacks Ack1(Ydl203c), an uncharacterized SEL-1 domain-containing protein that we find modulates pathway activity. Epistasis analysis places Ack1 upstream of Pkc1 in the CWI pathway and dependent on the upstream Rho1 GTP exchange factors Rom2 and Tus1. Overall, the synthetic genetic network around
PKC1
directly and efficiently identifies known and novel components of PKC signaling in yeast.
The Cell Wall of the Human Pathogen
Candida glabrata
: Differential Incorporation of Novel Adhesin-Like Wall Proteins
Authors: de Groot Piet W. J. , Kraneveld Eefje A. , Yin Qing Yuan , Dekker Henk L. , Groß Uwe , Crielaard Wim , de Koster Chris G. , Bader Oliver , Klis Frans M. , Weig Michael 
Published: September 2008

ABSTRACT

The cell wall of the human pathogen
Candida glabrata
governs initial host-pathogen interactions that underlie the establishment of fungal infections. With the aim of identifying species-specific features that may directly relate to its virulence, we have investigated the cell wall of
C. glabrata
using a multidisciplinary approach that combines microscopy imaging, biochemical studies, bioinformatics, and tandem mass spectrometry. Electron microscopy revealed a bilayered wall structure in which the outer layer is packed with mannoproteins. Biochemical studies showed that
C. glabrata
walls incorporate 50% more protein than
Saccharomyces cerevisiae
walls and, consistent with this, have a higher mannose/glucose ratio. Evidence is presented that
C. glabrata
walls contain glycosylphosphatidylinositol (GPI) proteins, covalently bound to the wall 1,6-β-glucan, as well as proteins linked through a mild-alkali-sensitive linkage to 1,3-β-glucan. A comprehensive genome-wide in silico inspection showed that in comparison to other fungi,
C. glabrata
contains an exceptionally large number, 67, of genes encoding adhesin-like GPI proteins. Phylogenetically these adhesin-like proteins form different clusters, one of which is the lectin-like
EPA
family. Mass spectrometric analysis identified 23 cell wall proteins, including 4 novel adhesin-like proteins, Awp1/2/3/4, and Epa6, which is involved in adherence to human epithelia and biofilm formation. Importantly, the presence of adhesin-like proteins in the wall depended on the growth stage and on the genetic background used, and this was reflected in alterations in adhesion capacity and cell surface hydrophobicity. We propose that the large repertoire of adhesin(-like) genes of
C. glabrata
contributes to its adaptability and virulence.
Nonapoptotic Death of
Saccharomyces cerevisiae
Cells That Is Stimulated by Hsp90 and Inhibited by Calcineurin and Cmk2 in Response to Endoplasmic Reticulum Stresses
Authors: Dudgeon Drew D. , Zhang Nannan , Ositelu Olufisayo O. , Kim Hyemin , Cunningham Kyle W. 
Published: September 2008

ABSTRACT

Endoplasmic reticulum (ER) stress can trigger apoptosis and necrosis in many types of mammalian cells. Previous studies in yeast found little or no cell death in response to the ER stressor tunicamycin, but a recent study suggested widespread apoptosis-like death. Here we show that wild-type laboratory
Saccharomyces cerevisiae
cells responding to tunicamycin die by nonapoptotic mechanisms in low-osmolyte culture media and survive for long periods of time in standard synthetic media. Survival requires calcineurin, a Ca
2+
/calmodulin-dependent protein phosphatase, but none of its known targets. The Ca
2+
/calmodulin-dependent protein kinase Cmk2 was identified as an indirect target of calcineurin that suppresses death of calcineurin-deficient cells. Death of Cmk2- and/or calcineurin-deficient
S. cerevisiae
cells was preceded by accumulation of reactive oxygen species but was not associated with hallmarks of apoptosis and was not dependent on Mca1, Aif1, Nuc1, or other factors implicated in apoptosis-like death. Cmk2 and calcineurin also independently suppressed the death of
S. cerevisiae
cells responding to dithiothreitol or miconazole, a common azole-class antifungal drug. Though inhibitors of Hsp90 have been shown to diminish calcineurin signaling in
S. cerevisiae
and to synergistically inhibit growth in combination with azoles, they did not stimulate death of
S. cerevisiae
cells in combination with miconazole or tunicamycin, and instead they prevented the death of calcineurin- and Cmk2-deficient cells. These findings reveal a novel prodeath role for Hsp90 and antideath roles for calcineurin and Cmk2 that extend the life span of
S. cerevisiae
cells responding to both natural and clinical antifungal compounds.
SigF Controls Carotenoid Pigment Production and Affects Transformation Efficiency and Hydrogen Peroxide Sensitivity in
Mycobacterium smegmatis
Authors: Provvedi Roberta , Kocíncová Dana , Donà Valentina , Euphrasie Daniel , Daffé Mamadou , Etienne Gilles , Manganelli Riccardo , Reyrat Jean-Marc 
Published: September 2008

ABSTRACT

Carotenoids are complex lipids that are known for acting against photodynamic injury and free radicals. We demonstrate here that σ
F
is required for carotenoid pigment production in
Mycobacterium smegmatis
. We further show that a
sigF
mutant exhibits a transformation efficiency 10
4
-fold higher than that of the parental strain, suggesting that σ
F
regulates the production of components affecting cell wall permeability. In addition, a
sigF
mutant showed an increased sensitivity to hydrogen peroxide. An in silico search of the
M. smegmatis
genome identified a number of SigF consensus sites, including sites upstream of the carotenoid synthesis locus, which explains its SigF regulation.
Mechanism of 4-Nitrophenol Oxidation in
Rhodococcus
sp. Strain PN1: Characterization of the Two-Component 4-Nitrophenol Hydroxylase and Regulation of Its Expression
Authors: Takeo Masahiro , Murakami Masumi , Niihara Sanae , Yamamoto Kenta , Nishimura Munehiro , Kato Dai-ichiro , Negoro Seiji 
Published: September 2008

ABSTRACT

4-Nitrophenol (4-NP) is a toxic product of the hydrolysis of organophosphorus pesticides such as parathion in soil.
Rhodococcus
sp. strain PN1 degrades 4-NP via 4-nitrocatechol (4-NC) for use as the sole carbon, nitrogen, and energy source. A 5-kb EcoRI DNA fragment previously cloned from PN1 contained a gene cluster (
nphRA1A2
) involved in 4-NP oxidation. From sequence analysis, this gene cluster is expected to encode an AraC/XylS family regulatory protein (NphR) and a two-component 4-NP hydroxylase (NphA1 and NphA2). A transcriptional assay in a
Rhodococcus
strain revealed that the transcription of
nphA1
is induced by only 4-NP (of several phenolic compounds tested) in the presence of
nphR
, which is constitutively expressed. Disruption of
nphR
abolished transcriptional activity, suggesting that
nphR
encodes a positive regulatory protein. The two proteins of the 4-NP hydroxylase, NphA1 and NphA2, were independently expressed in
Escherichia coli
and purified by ion-exchange chromatography or affinity chromatography. The purified NphA2 reduced flavin adenine dinucleotide (FAD) with the concomitant oxidation of NADH, while the purified NphA1 oxidized 4-NP into 4-NC almost quantitatively in the presence of FAD, NADH, and NphA2. This functional analysis, in addition to the sequence analysis, revealed that this enzyme system belongs to the two-component flavin-diffusible monooxygenase family. The 4-NP hydroxylase showed comparable oxidation activities for phenol and 4-chlorophenol to that for 4-NP and weaker activities for 3-NP and 4-NC.
Passage through
Tetrahymena tropicalis
Triggers a Rapid Morphological Differentiation in
Legionella pneumophila
Authors: Faulkner Gary , Berk Sharon G. , Garduño Elizabeth , Ortiz-Jiménez Marco A. , Garduño Rafael A. 
Published: September 2008

ABSTRACT

The intracellular bacterial pathogen
Legionella pneumophila
follows a developmental cycle in which replicative forms (RFs) differentiate into infectious stationary-phase forms (SPFs) in vitro and in vivo into highly infectious mature intracellular forms (MIFs). The potential relationships between SPFs and MIFs remain uncharacterized. Previously we determined that
L. pneumophila
survives, but does not replicate, while it transiently resides (for 1 to 2 h) in food vacuoles of the freshwater ciliate
Tetrahymena tropicalis
before being expelled as legionellae-laden pellets. We report here that SPFs have the ability to rapidly (<1 h) and directly (in the absence of bacterial replication) differentiate into MIFs while in transit through
T. tropicalis
, indicating that SPFs and MIFs constitute a differentiation continuum. Mutant RFs lacking the sigma factor gene
rpoS
, or the response regulator gene
letA
, were unable to produce normal SPFs in vitro and did not fully differentiate into MIFs in vivo, further supporting the existence of a common mechanism of differentiation shared by SPFs and MIFs. Mutants with a defective Dot/Icm system morphologically differentiated into MIFs while in transit through
T. tropicalis
. Therefore,
T. tropicalis
has allowed us to unequivocally conclude that SPFs can directly differentiate into MIFs and that the Dot/Icm system is not required for differentiation, two events that could not be experimentally addressed before. The
Tetrahymena
model can now be exploited to study the signals that trigger MIF development in vivo and is the only replication-independent model reported to date that allows the differentiation of Dot/Icm mutants into MIFs.
Assemblies of DivIVA Mark Sites for Hyphal Branching and Can Establish New Zones of Cell Wall Growth inStreptomyces coelicolor
Authors: Hempel Antje Marie , Wang Sheng-bing , Letek Michal , Gil José A. , Flärdh Klas 
Published: September 2008

ABSTRACTTime-lapse imaging ofStreptomyceshyphae revealed foci of the essential protein DivIVA at sites where lateral branches will emerge. Overexpression experiments showed that DivIVA foci can trigger establishment of new zones of cell wall assembly, suggesting a key role of DivIVA in directing peptidoglycan synthesis and cell shape inStreptomyces.
MacAB Is Involved in the Secretion ofEscherichia coliHeat-Stable Enterotoxin II
Authors: Yamanaka Hiroyasu , Kobayashi Hidetomo , Takahashi Eizo , Okamoto Keinosuke 
Published: September 2008

ABSTRACTThe heat-stable enterotoxin (ST) produced by enterotoxigenicEscherichia coliis an extracellular peptide toxin that evokes watery diarrhea in the host. Two types of STs, STI and STII, have been found. Both STs are synthesized as precursor proteins and are then converted to the active forms with intramolecular disulfide bonds after being released into the periplasm. The active STs are finally translocated across the outer membrane through a tunnel made by TolC. However, it is unclear how the active STs formed in the periplasm are led to the TolC channel. Several transporters in the inner membrane and their periplasmic accessory proteins are known to combine with TolC and form a tripartite transport system. We therefore expect such transporters to also act as a partner with TolC to export STs from the periplasm to the exterior. In this study, we carried out pulse-chase experiments usingE. coliBL21(DE3) mutants in which various transporter genes (acrAB, acrEF, emrAB, emrKY, mdtEF, macAB, andyojHI) had been knocked out and analyzed the secretion of STs in those strains. The results revealed that the extracellular secretion of STII was largely decreased in themacABmutant and the toxin molecules were accumulated in the periplasm, although the secretion of STI was not affected in any mutant used in this study. The periplasmic stagnation of STII in themacABmutant was restored by the introduction of pACYC184, containing themacABgene, into the cell. These results indicate that MacAB, an ATP-binding cassette transporter of MacB and its accessory protein, MacA, participates in the translocation of STII from the periplasm to the exterior. Since it has been reported that MacAB cooperates with TolC, we propose that the MacAB-TolC system captures the periplasmic STII molecules and exports the toxin molecules to the exterior.
Role of Two NtcA-Binding Sites in the Complex
ntcA
Gene Promoter of the Heterocyst-Forming Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Olmedo-Verd Elvira , Valladares Ana , Flores Enrique , Herrero Antonia , Muro-Pastor Alicia M. 
Published: September 2008

ABSTRACT

Anabaena
sp. strain PCC 7120 is a filamentous cyanobacterium that fixes N
2
in specialized cells called heterocysts, which differentiate from vegetative cells in a process that requires the nitrogen control transcription factor NtcA. 2-Oxoglutarate-stimulated binding of purified NtcA to wild-type and modified versions of the
ntcA
gene promoter from
Anabaena
sp. was analyzed by mobility shift and DNase I footprinting assays, and the role of NtcA-binding sites in the expression of the
ntcA
gene during heterocyst differentiation was studied in vivo by using an
ntcA-gfp
translational fusion and primer extension analysis. Mutation of neither of the two identified NtcA-binding sites eliminated localized expression of
ntcA
in proheterocysts, but mutation of both sites led to very low, nonlocalized expression.
YspM, a Newly Identified Ysa Type III Secreted Protein ofYersinia enterocolitica
Authors: Witowski Sarah E. , Walker Kimberly A. , Miller Virginia L. 
Published: September 2008

ABSTRACTYersinia enterocoliticahas three type three secretion systems, the flagellar, the plasmid Ysc type III secretion system (T3SS), and the chromosomal Ysa T3SS. The Ysc T3SS, through the proteins it secretes (Yops), prevents phagocytosis ofY. enterocoliticaand is required for disease processes in the mouse host. Recent data demonstrate a role for the Ysa T3SS during initial colonization of the mouse via secretion of Ysps (Yersiniasecreted proteins). This work characterizes the discovery of a newly identified Ysa type III secreted protein, YspM. Expression ofyspMis regulated by temperature, NaCl concentration, and other known regulators of theysasystem. In addition, YspM is translocated into host cells via the Ysa T3SS. YspM is homologous to proteins classified as GDSL bacterial lipases, which possess a catalytic triad of amino acids (Ser, Asp, and His) located in three of five blocks of amino acid identity. Sequence analysis of the JB580v strain ofY. enterocoliticashows that, due to a premature stop codon, it no longer encodes the fifth block of amino acid identity containing the predicted catalytic histidine. However, seven other biotype 1B strains sequenced did possess the domain. A functional difference between the forms was revealed when YspM was expressed inSaccharomyces cerevisiae. Yeast growth was uninhibited when YspM from JB580v was expressed but greatly inhibited when YspM from Y295 (YspMY295) was expressed. Site-directed mutagenesis of the histidine of YspMY295ablated the toxic effects. These results indicate that YspM is secreted by the Ysa T3SS and that, possibly due to lipase activity, it targets eukaryotic cellular component(s).
Kinetic Analysis of Growth Rate, ATP, and Pigmentation Suggests an Energy-Spilling Function for the Pigment Prodigiosin ofSerratia marcescens
Authors: Haddix Pryce L. , Jones Sarah , Patel Pratik , Burnham Sarah , Knights Kaori , Powell Joan N. , LaForm Amber 
Published: September 2008

ABSTRACTSerratia marcescensis a gram-negative environmental bacterium and opportunistic pathogen.S. marcescensexpresses prodigiosin, a bright red and cell-associated pigment which has no known biological function for producing cells. We present here a kinetic model relating cell, ATP, and prodigiosin concentration changes forS. marcescensduring cultivation in batch culture. Cells were grown in a variety of complex broth media at temperatures which either promoted or essentially prevented pigmentation. High growth rates were accompanied by large decreases in cellular prodigiosin concentration; low growth rates were associated with rapid pigmentation. Prodigiosin was induced most strongly during limited growth as the population transitioned to stationary phase, suggesting a negative effect of this pigment on biomass production. Mathematically, the combined rate of formation of biomass and bioenergy (as ATP) was shown to be equivalent to the rate of prodigiosin production. Studies with cyanide inhibition of both oxidative phosphorylation and pigment production indicated that rates of biomass and net ATP synthesis were actually higher in the presence of cyanide, further suggesting a negative regulatory role for prodigiosin in cell and energy production under aerobic growth conditions. Considered in the context of the literature, these results suggest that prodigiosin reduces ATP production by a process termed energy spilling. This process may protect the cell by limiting production of reactive oxygen compounds. Other possible functions for prodigiosin as a mediator of cell death at population stationary phase are discussed.