bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reduced Folate Supply as a Key to Enhanced
l
-Serine Production by
Corynebacterium glutamicum
Authors: Stolz Michael , Peters-Wendisch Petra , Etterich Helga , Gerharz Tanja , Faurie Robert , Sahm Hermann , Fersterra Holger , Eggeling Lothar 
Published: December 2006

ABSTRACT

The amino acid
l
-serine is required for pharmaceutical purposes, and the availability of a sugar-based microbial process for its production is desirable. However, a number of intracellular utilization routes prevent overproduction of
l
-serine, with the essential serine hydroxymethyltransferase (SHMT) (
glyA
) probably occupying a key position. We found that constructs of
Corynebacterium glutamicum
strains where chromosomal
glyA
expression is dependent on P

tac

and
lacI
Q
are unstable, acquiring mutations in
lacI
Q
, for instance. To overcome the inconvenient
glyA
expression control, we instead considered controlling SHMT activity by the availability of 5,6,7,8-tetrahydrofolate (THF). The
pabAB
and
pabC
genes of THF synthesis were identified and deleted in
C. glutamicum
, and the resulting strains were shown to require folate or 4-aminobenzoate for growth. Whereas the
C. glutamicum
Δ
sdaA
strain (p
serACB
) accumulates only traces of
l
-serine, with the
C. glutamicum
Δ
pabABC
Δ
sdaA
strain (p
serACB
),
l
-serine accumulation and growth responded in a dose-dependent manner to an external folate supply. At 0.1 mM folate, 81 mM
l
-serine accumulated. In a 20-liter controlled fed-batch culture, a 345 mM
l
-serine accumulation was achieved. Thus, an efficient and highly competitive process for microbial
l
-serine production is available.
Microbial Community Structure and Dynamics of Dark Fire-Cured Tobacco Fermentation
Authors: Di Giacomo Michele , Paolino Marianna , Silvestro Daniele , Vigliotta Giovanni , Imperi Francesco , Visca Paolo , Alifano Pietro , Parente Dino 
Published: December 2006

ABSTRACT

The Italian Toscano cigar production includes a fermentation step that starts when dark fire-cured tobacco leaves are moistened and mixed with ca. 20% prefermented tobacco to form a 500-kg bulk. The dynamics of the process, lasting ca. 18 days, has never been investigated in detail, and limited information is available on microbiota involved. Here we show that Toscano fermentation is invariably associated with the following: (i) an increase in temperature, pH, and total microbial population; (ii) a decrease in reducing sugars, citric and malic acids, and nitrate content; and (iii) an increase in oxalic acid, nitrite, and tobacco-specific nitrosamine content. The microbial community structure and dynamics were investigated by culture-based and culture-independent approaches, including denaturing gradient gel electrophoresis and single-strand conformational polymorphism. Results demonstrate that fermentation is assisted by a complex microbial community, changing in structure and composition during the process. During the early phase, the moderately acidic and mesophilic environment supports the rapid growth of a yeast population predominated by
Debaryomyces hansenii
. At this stage,
Staphylococcaceae
(
Jeotgalicoccus
and
Staphylococcus
) and
Lactobacillales
(
Aerococcus
,
Lactobacillus
, and
Weissella
) are the most commonly detected bacteria. When temperature and pH increase, endospore-forming low-G+C content gram-positive bacilli (
Bacillus
spp.) become evident. This leads to a further pH increase and promotes growth of moderately halotolerant and alkaliphilic
Actinomycetales
(
Corynebacterium
and
Yania
) during the late phase. To postulate a functional role for individual microbial species assisting the fermentation process, a preliminary physiological and biochemical characterization of representative isolates was performed.
Prenylation of
Saccharomyces cerevisiae
Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length
Authors: Grabińska Kariona A. , Magnelli Paula , Robbins Phillips W. 
Published: December 2006

ABSTRACT

Chs4p (Cal2/Csd4/Skt5) was identified as a protein factor physically interacting with Chs3p, the catalytic subunit of chitin synthase III (CSIII), and is indispensable for its enzymatic activity in vivo. Chs4p contains a putative farnesyl attachment site at the C-terminal end (CVIM motif) conserved in Chs4p of
Saccharomyces cerevisiae
and other fungi. Several previous reports questioned the role of Chs4p prenylation in chitin biosynthesis. In this study we reinvestigated the function of Chs4p prenylation. We provide evidence that Chs4p is farnesylated by showing that purified Chs4p is recognized by anti-farnesyl antibody and is a substrate for farnesyl transferase (FTase) in vitro and that inactivation of FTase increases the amount of unmodified Chs4p in yeast cells. We demonstrate that abolition of Chs4p prenylation causes a ∼60% decrease in CSIII activity, which is correlated with a ∼30% decrease in chitin content and with increased resistance to the chitin binding compound calcofluor white. Furthermore, we show that lack of Chs4p prenylation decreases the average chain length of the chitin polymer. Prenylation of Chs4p, however, is not a factor that mediates plasma membrane association of the protein. Our results provide evidence that the prenyl moiety attached to Chs4p is a factor modulating the activity of CSIII both in vivo and in vitro.
Family Shuffling of Soil DNA To Change the Regiospecificity of
Burkholderia xenovorans
LB400 Biphenyl Dioxygenase
Authors: Vézina Julie , Barriault Diane , Sylvestre Michel 
Published: December 2006

ABSTRACT

Previous work has shown that the C-terminal portion of BphA, especially two amino acid segments designated region III and region IV, influence the regiospecificity of the biphenyl dioxygenase (BPDO) toward 2,2′-dichlorobiphenyl (2,2′-CB). In this work, we evolved BPDO by shuffling
bphA
genes amplified from polychlorinated biphenyl-contaminated soil DNA. Sets of approximately 1-kb DNA fragments were amplified with degenerate primers designed to amplify the C-terminal portion of
bphA
. These fragments were shuffled, and the resulting library was used to replace the corresponding fragment of
Burkholderia xenovorans
LB400
bphA.
Variants were screened for their ability to oxygenate 2,2′-CB onto carbons 5 and 6, which are positions that LB400 BPDO is unable to attack. Variants S100, S149, and S151 were obtained and exhibited this feature. Variant S100 BPDO produced exclusively
cis
-5,6-dihydro-5,6-dihydroxy-2,2′-dichlorobiphenyl from 2,2′-CB. Moreover, unlike LB400 BPDO, S100 BphA catalyzed the oxygenation of 2,2′,3,3′-tetrachlorobiphenyl onto carbons 5 and 6 exclusively and it was unable to oxygenate 2,2′,5,5′-tetrachlorobiphenyl. Based on oxygen consumption measurements, variant S100 oxygenated 2,2′-CB at a rate of 16 ± 1 nmol min
−1
per nmol enzyme, which was similar to the value observed for LB400 BPDO.
cis
-5,6-Dihydro-5,6-dihydroxy-2,2′-dichlorobiphenyl was further oxidized by 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) and 2,3-dihydroxybiphenyl dioxygenase (BphC). Variant S100 was, in addition, able to oxygenate benzene, toluene, and ethyl benzene. Sequence analysis identified amino acid residues M
237
S
238
and S
283
outside regions III and IV that influence the activity toward doubly
ortho
-substituted chlorobiphenyls.
Proteome and Differential Expression Analysis of Membrane and Cytosolic Proteins from
Mycobacterium avium
subsp.
paratuberculosis
Strains K-10 and 187
Authors: Radosevich Thomas J. , Reinhardt Timothy A. , Lippolis John D. , Bannantine John P. , Stabel Judith R. 
Published: December 2006

ABSTRACT

Little is known of protein expression in
Mycobacterium avium
subsp.
paratuberculosis
and how this contributes to pathogenesis. In the present study, proteins from both membranes and cytosol were prepared from two strains of
M. avium
subsp.
paratuberculosis
, i.e., laboratory-adapted strain K-10 and a recent isolate, strain 187, obtained from a cow exhibiting clinical signs of Johne's disease. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytosol and membrane proteins from K-10 and 187 showed marked differences in protein expression. Relative levels of protein expression from both
M. avium
subsp.
paratuberculosis
strains were measured by using amine-reactive isobaric tagging reagents (iTRAQ) and tandem mass spectroscopy. Protein identification and relative expression data were obtained for 874 membrane and cytosolic proteins from the
M. avium
subsp.
paratuberculosis
proteome. These data showed a number of significant differences in protein expression between strain K-10 and clinical isolate 187. Examples of proteins expressed at higher levels in clinical isolate 187 compared to strain K-10 are AtpC, RpoA, and several proteins involved in fatty acid biosynthesis. In contrast, proteins such as AhpC and several proteins involved in nitrogen metabolism were expressed at higher levels in strain K-10 compared to strain 187. These data may provide insights into the proteins whose expression is important in natural infection but are modified once
M. avium
subsp.
paratuberculosis
is adapted to laboratory cultivation. Results from these studies will provide tools for developing a better understanding of
M. avium
subsp.
paratuberculosis
infection in the host and offer potential as diagnostic reagents and vaccine candidates.
Expression of the Major Porin Gene
mspA
Is Regulated in
Mycobacterium smegmatis
Authors: Hillmann Dietmar , Eschenbacher Iris , Thiel Anja , Niederweis Michael 
Published: December 2006

ABSTRACT

MspA is the major porin of
Mycobacterium smegmatis
and is important for diffusion of small and hydrophilic solutes across its unique outer membrane. The start point of transcription of the
mspA
gene was mapped by primer extension and S1 nuclease experiments. The main promoter driving transcription of
mspA
was identified by single point mutations in
lacZ
fusions and resembled σ
A
promoters of
M. smegmatis
. However, a 500-bp upstream fragment including P

mspA

in a transcriptional fusion with
lacZ
yielded only low β-galactosidase activity, whereas activity increased 12-fold with a 700-bp fragment. Activation of P

mspA

by the 200-bp element was almost eliminated by increasing the distance by 14 bp, indicating binding of an activator protein. The chromosomal
mspA
transcript had a size of 900 bases and was very stable with a half-life of 6 minutes, whereas the stabilities of episomal
mspA
transcripts with three other 5′ untranslated region (UTRs) were three- to sixfold reduced, indicating a stabilizing role of the native 5′ UTR of
mspA
. Northern blot experiments revealed that the amount of
mspA
mRNA was increased under nitrogen limitation but reduced under carbon and phosphate limitation at 42°C in stationary phase in the presence of 0.5 M sodium chloride, 18 mM hydrogen peroxide, and 10% ethanol and at acidic pH. These results show for the first time that
M. smegmatis
regulates porin gene expression to optimize uptake of certain nutrients and to protect itself from toxic solutes.
A Second Galacturonic Acid Transferase Is Required for Core Lipopolysaccharide Biosynthesis and Complete Capsule Association with the Cell Surface in
Klebsiella pneumoniae
Authors: Fresno Sandra , Jiménez Natalia , Canals Rocío , Merino Susana , Corsaro Maria Michela , Lanzetta Rosa , Parrilli Michelangelo , Pieretti Giuseppina , Regué Miguel , Tomás Juan M. 
Published: December 2006

ABSTRACT

The core lipopolysaccharide (LPS) of
Klebsiella pneumoniae
contains two galacturonic acid (GalA) residues, but only one GalA transferase (WabG) has been identified. Data from chemical and structural analysis of LPS isolated from a
wabO
mutant show the absence of the inner core β-GalA residue linked to
l
-
glycero
-
d
-
manno
-heptose III (
l
,
d
-Hep III). An in vitro assay demonstrates that the purified WabO is able to catalyze the transfer of GalA from UDP-GalA to the acceptor LPS isolated from the
wabO
mutant, but not to LPS isolated from
waaQ
mutant (deficient in
l
,
d
-Hep III). The absence of this inner core β-GalA residue results in a decrease in virulence in a capsule-dependent experimental mouse pneumonia model. In addition, this mutation leads to a strong reduction in cell-bound capsule. Interestingly, a K66
Klebsiella
strain (natural isolate) without a functional
wabO
gene shows reduced levels of cell-bound capsule in comparison to those of other K66 strains. Thus, the WabO enzyme plays an important role in core LPS biosynthesis and determines the level of cell-bound capsule in
Klebsiella pneumoniae
.
A Stochastic Mechanism for Biofilm Formation by
Mycoplasma pulmonis
Authors: Simmons Warren L. , Bolland Jeffrey R. , Daubenspeck James M. , Dybvig Kevin 
Published: December 2006

ABSTRACT

Bacterial biofilms are communities of bacteria that are enclosed in an extracellular matrix. Within a biofilm the bacteria are protected from antimicrobials, environmental stresses, and immune responses from the host. Biofilms are often believed to have a highly developed organization that is derived from differential regulation of the genes that direct the synthesis of the extracellular matrix and the attachment to surfaces. The mycoplasmas have the smallest of the prokaryotic genomes and apparently lack complex gene-regulatory systems. We examined biofilm formation by
Mycoplasma pulmonis
and found it to be dependent on the length of the tandem repeat region of the variable surface antigen (Vsa) protein. Mycoplasmas that produced a short Vsa protein with few tandem repeats formed biofilms that attached to polystyrene and glass. Mycoplasmas that produced a long Vsa protein with many tandem repeats formed microcolonies that floated freely in the medium. The biofilms and the microcolonies contained an extracellular matrix which contained Vsa protein, lipid, DNA, and saccharide. As variation in the number of Vsa tandem repeats occurs by slipped-strand mispairing, the ability of the mycoplasmas to form a biofilm switches stochastically.
Anionic Lipids Enriched at the ExPortal of
Streptococcus pyogenes
Authors: Rosch Jason W. , Hsu Fong Fu , Caparon Michael G. 
Published: December 2006

ABSTRACT

The ExPortal of
Streptococcus pyogenes
is a membrane microdomain dedicated to the secretion and folding of proteins. We investigated the lipid composition of the ExPortal by examining the distribution of anionic membrane phospholipids. Staining with 10-
N
-nonyl-acridine orange revealed a single microdomain enriched with an anionic phospholipid whose staining characteristics and behavior in a cardiolipin-deficient mutant were characteristic of phosphatidylglycerol. Furthermore, the location of the microdomain corresponded to the site of active protein secretion at the ExPortal. These results indicate that the ExPortal is an asymmetric lipid microdomain, whose enriched content of anionic phospholipids may play an important role in ExPortal organization and protein trafficking.
Role of Phosphoenolpyruvate in the NADP-Isocitrate Dehydrogenase and Isocitrate Lyase Reaction in
Escherichia coli
Authors: Ogawa Tadashi , Murakami Keiko , Mori Hirotada , Ishii Nobuyoshi , Tomita Masaru , Yoshin Masataka 
Published: December 2006

ABSTRACT

Phosphoenolpyruvate inhibited
Escherichia coli
NADP-isocitrate dehydrogenase allosterically (

K
i

of 0.31 mM) and isocitrate lyase uncompetitively (

K
i

′ of 0.893 mM). Phosphoenolpyruvate enhances the uncompetitive inhibition of isocitrate lyase by increasing isocitrate, which protects isocitrate dehydrogenase from the inhibition, and contributes to the control through the tricarboxylic acid cycle and glyoxylate shunt.
Haemodynamic changes induced by submaximal exercise before a dive and its consequences on bubble formation
Authors: Blatteau Jean-Eric , Boussuges Alain , Gempp Emmanuel , Pontier Jean-Michel , Castagna Olivier , Robinet Claude , Galland Francois-Michel , Bourdon Lionel 
Published: December 2006

Objectives:To evaluate the effects of a submaximal exercise performed 2 h before a simulated dive on bubble formation and to observe the haemodynamic changes and their influence on bubble formation.Participants and methods:16 trained divers were compressed in a hyperbaric chamber to 400 kPa for 30 min and decompressed at a rate of 100 kPa/min with a 9 min stop at 130 kPa (French Navy MN90 procedure). Each diver performed two dives 3 days apart, one without exercise and one with exercise before the dive. All participants performed a 40 min constant-load submaximal and calibrated exercise, which consisted of outdoor running 2 h before the dive. Circulating bubbles were detected with a precordial Doppler at 30, 60 and 90 min after surfacing. Haemodynamic changes were evaluated with Doppler echocardiography.Results:A single bout of strenuous exercise 2 h before a simulated dive significantly reduced circulating bubbles. Post-exercise hypotension (PEH) was observed after exercise with reductions in diastolic and mean blood pressure (DBP and MBP), but total peripheral resistance was unchanged. Stroke volume was reduced, whereas cardiac output was unchanged. Simulated diving caused a similar reduction in cardiac output independent of pre-dive exercise, suggesting that pre-dive exercise only changed DBP and MBP caused by reduced stroke volume.Conclusion:A single bout of strenuous exercise 2 h before a dive significantly reduced the number of bubbles in the right heart of divers and protected them from decompression sickness. Declining stroke volume and moderate dehydration induced by a pre-dive exercise might influence inert gas load and bubble formation.
Injuries in amateur horse racing (point to point racing) in Great Britain and Ireland during 1993–2006
Authors: Balendra Ganesh , Turner Michael , McCrory Paul , Halley Walter 
Published: December 2006

Objectives:To provide a breakdown of injury incidence from amateur jump racing (also known as point to point racing) in Great Britain and Ireland during 1993–2006 and to compare the injury epidemiology with professional horse racing in Great Britain, Ireland and France.Design:Retrospective review.Setting:Great Britain and Ireland.Participants:Amateur jockeys.Main outcome measures:Injury rates.Results:Injury data suggest that point to point racing is more dangerous from an injury point of view than professional jump racing, which has previously been shown to be more dangerous than flat racing. Amateur jockeys have more falls than their professional counterparts, and this in turn puts them at greater risk of sustaining more serious injuries.Conclusions:Amateur (point to point) jockeys represent a sporting population that previously has been little studied. They represent a group at high risk of injury, and hence formal injury surveillance tracking and counter measures for injury prevention are recommended.
Ccr5 But Not Ccr1 Deficiency Reduces Development of Diet-Induced Atherosclerosis in Mice
Authors: Braunersreuther Vincent , Zernecke Alma , Arnaud Claire , Liehn Elisa A. , Steffens Sabine , Shagdarsuren Erdenechimeg , Bidzhekov Kiril , Burger Fabienne , Pelli Graziano , Luckow Bruno , Mach François , Weber Christian 
Published: December 2006

Objective—Chemokines and their receptors are crucially involved in the development of atherosclerotic lesions by directing monocyte and T cell recruitment. The CC-chemokine receptors 1 (CCR1) and 5 (CCR5) expressed on these cells bind chemokines implicated in atherosclerosis, namely CCL5/RANTES. Although general blockade of CCL5 receptors reduces atherosclerosis, specific roles of CCR1 and CCR5 have not been unequivocally determined.Methods and Results—We provide two independent lines of investigation to dissect the effects of Ccr1 and Ccr5 deletion in apolipoprotein E–deficient (ApoE−/−) mice in a collaboration between Aachen/Germany and Geneva/Switzerland. Different strains of ApoE−/−Ccr5−/−mice, ApoE−/−Ccr1−/−mice or respective littermates, were fed a high-fat diet for 10 to 12 weeks. Plaque areas were quantified in the aortic roots and thoracoabdominal aortas. Concordantly, both laboratories found that lesion formation was reduced in ApoE−/−Ccr5−/−mice. Plaque quality and immune cells were assessed by immunohistochemistry or mRNA analysis. Whereas lesional macrophage content, aortic CD4, and Th1-related Tim3 expression were reduced, smooth muscle cell (SMC) content and expression of interleukin-10 in plaques, lesional SMCs, and splenocytes were elevated. Protection against lesion formation by Ccr5 deficiency was sustained over 22 weeks of high-fat diet or over 26 weeks of chow diet. Conversely, plaque area, T cell, and interferon-γ content were increased in ApoE−/−Ccr1−/−mice.Conclusion—Genetic deletion of Ccr5 but not Ccr1 in ApoE−/−mice protects from diet-induced atherosclerosis, associated with a more stable plaque phenotype, reduced mononuclear cell infiltration, Th1-type immune responses, and increased interleukin-10 expression. This corroborates CCR5 as a promising therapeutic target.
Human herpesvirus 8 acute infection of endothelial cells induces monocyte chemoattractant protein 1–dependent capillary-like structure formation: role of the IKK/NF-κB pathway
Authors: Caselli Elisabetta , Fiorentini Simona , Amici Carla , Di Luca Dario , Caruso Arnaldo , Santoro M. Gabriella 
Published: December 2006

AbstractHuman herpesvirus 8 (HHV-8) is considered the causative agent of Kaposi sarcoma, a highly vascularized neoplasm characterized by spindle-shaped cells of endothelial origin and inflammatory cell infiltration. The cell transforming ability of HHV-8 has been associated with the activation of NF-κB, a nuclear factor playing a pivotal role in promoting inflammation and cell proliferation; however, little is known about NF-κB activation during acute HHV-8 infection. In the present study, we used a recently established in vitro model of HHV-8 acute productive infection in endothelial cells to investigate the effect of HHV-8 on NF-κB activity and function. HHV-8 rapidly and potently induced NF-κB activity in endothelial cells via stimulation of the IκB kinase (IKK). Following IKK activation, HHV-8 selectively triggered the production of high levels of monocyte chemoattractant protein 1 (MCP-1), whereas it did not affect the expression of other NF-κB–dependent proinflammatory proteins, including TNF-α, IL-8, and RANTES. Deletion of NF-κB–binding sites in the MCP-1 enhancer resulted in significant inhibition of HHV-8–induced transcription. Furthermore, MCP-1 production was accompanied by virus-induced capillary-like structure formation at early stages of infection. The results suggest that HHV-8–induced MCP-1 may play an important role in promoting inflammation and pathogenic angiogenesis typical of HHV-8–associated lesions.