bluuebunny/crossref_metadata_2025_split huggingface dataset:
Engineering of
Saccharomyces cerevisiae
for Efficient Anaerobic Alcoholic Fermentation of
l
-Arabinose
Authors: Wisselink H. Wouter , Toirkens Maurice J. , del Rosario Franco Berriel M. , Winkler Aaron A. , van Dijken Johannes P. , Pronk Jack T. , van Maris Antonius J. A. 
Published: June 2007

ABSTRACT

For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as
l
-arabinose, is required. Wild-type strains of
Saccharomyces cerevisiae
, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of
l
-arabinose into ethanol by engineered
S. cerevisiae
strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of
l
-arabinose by an engineered
S. cerevisiae
strain. This fermentation was achieved by combining the expression of the structural genes for the
l
-arabinose utilization pathway of
Lactobacillus plantarum
, the overexpression of the
S. cerevisiae
genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting
S. cerevisiae
strain exhibited high rates of arabinose consumption (0.70 g h
−1
g [dry weight]
−1
) and ethanol production (0.29 g h
−1
g [dry weight]
−1
) and a high ethanol yield (0.43 g g
−1
) during anaerobic growth on
l
-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.
Quantitative Analysis of Population Heterogeneity of the Adaptive Salt Stress Response and Growth Capacity of
Bacillus cereus
ATCC 14579
Authors: den Besten Heidy M. W. , Ingham Colin J. , van Hylckama Vlieg Johan E. T. , Beerthuyzen Marke M. , Zwietering Marcel H. , Abee Tjakko 
Published: June 2007

ABSTRACT

Bacterial populations can display heterogeneity with respect to both the adaptive stress response and growth capacity of individual cells. The growth dynamics of
Bacillus cereus
ATCC 14579 during mild and severe salt stress exposure were investigated for the population as a whole in liquid culture. To quantitatively assess the population heterogeneity of the stress response and growth capacity at a single-cell level, a direct imaging method was applied to monitor cells from the initial inoculum to the microcolony stage. Highly porous Anopore strips were used as a support for the culturing and imaging of microcolonies at different time points. The growth kinetics of cells grown in liquid culture were comparable to those of microcolonies grown upon Anopore strips, even in the presence of mild and severe salt stress. Exposure to mild salt stress resulted in growth that was characterized by a remarkably low variability of microcolony sizes, and the distributions of the log
10
-transformed microcolony areas could be fitted by the normal distribution. Under severe salt stress conditions, the microcolony sizes were highly heterogeneous, and this was apparently caused by the presence of both a nongrowing and growing population. After discriminating these two subpopulations, it was shown that the variability of microcolony sizes of the growing population was comparable to that of non-salt-stressed and mildly salt-stressed populations. Quantification of population heterogeneity during stress exposure may contribute to an optimized application of preservation factors for controlling growth of spoilage and pathogenic bacteria to ensure the quality and safety of minimally processed foods.
Agrobacterium tumefaciens fur
Has Important Physiological Roles in Iron and Manganese Homeostasis, the Oxidative Stress Response, and Full Virulence
Authors: Kitphati Worawan , Ngok-ngam Patchara , Suwanmaneerat Sukanya , Sukchawalit Rojana , Mongkolsuk Skorn 
Published: June 2007

ABSTRACT

In
Agrobacterium tumefaciens
, the balance between acquiring enough iron and avoiding iron-induced toxicity is regulated in part by Fur (ferric uptake regulator). A
fur
mutant was constructed to address the physiological role of the regulator. Atypically, the mutant did not show alterations in the levels of siderophore biosynthesis and the expression of iron transport genes. However, the
fur
mutant was more sensitive than the wild type to an iron chelator, 2,2′-dipyridyl, and was also more resistant to an iron-activated antibiotic, streptonigrin, suggesting that Fur has a role in regulating iron concentrations.
A. tumefaciens sitA
, the periplasmic binding protein of a putative ABC-type iron and manganese transport system (
sitABCD
), was strongly repressed by Mn
2+
and, to a lesser extent, by Fe
2+
, and this regulation was Fur dependent. Moreover, the
fur
mutant was more sensitive to manganese than the wild type. This was consistent with the fact that the
fur
mutant showed constitutive up-expression of the manganese uptake
sit
operon. Fur
At
showed a regulatory role under iron-limiting conditions. Furthermore, Fur has a role in determining oxidative resistance levels. The
fur
mutant was hypersensitive to hydrogen peroxide and had reduced catalase activity. The virulence assay showed that the
fur
mutant had a reduced ability to cause tumors on tobacco leaves compared to wild-type NTL4.
Isolation and Characterization of a Novel Haloacid Permease from
Burkholderia cepacia
MBA4
Authors: Yu Manda , Faan Yun-Wing , Chung Wilson Y. K. , Tsang Jimmy S. H. 
Published: June 2007

ABSTRACT

Burkholderia cepacia
MBA4 is a bacterium that can utilize 2-haloacids as carbon and energy sources for growth. It has been proposed that dehalogenase-associated permease mediates the uptake of haloacid. In this paper, we report the first cloning and characterization of such a haloacid permease. The structural gene, designated
deh4p
, was found 353 bases downstream of the dehalogenase gene
deh4a
. Quantitative analysis of the expression of
deh4p
showed that it was induced by monochloroacetate (MCA), to a level similar to the MCA-induced level of
deh4a
. The nucleotide sequence of
deh4p
was determined, and an open reading frame of 1,656 bp encoding a putative peptide of 552 amino acids was identified. Deh4p has a putative molecular weight of 59,414 and an isoelectric point of 9.88. Deh4p has the signatures of sugar transport proteins and integral membrane proteins of the major facilitator superfamily. Uptake of [
14
C]MCA into the cell was Deh4p dependent. Deh4p has apparent

K
m

s of 5.5 and 8.9 μM and
V
max
s of 9.1 and 23.1 nmol mg
−1
min
−1
for acetate and MCA, respectively. A mutant with a transposon-inactivated haloacid operon failed to grow on MCA even when
deh4a
was provided in
trans
.
NCgl2620 Encodes a Class II Polyphosphate Kinase in
Corynebacterium glutamicum
Authors: Lindner Steffen N. , Vidaurre Dolores , Willbold Sabine , Schoberth Siegfried M. , Wendisch Volker F. 
Published: June 2007

ABSTRACT

Corynebacterium glutamicum
is able to accumulate up to 600 mM cytosolic phosphorus in the form of polyphosphate (poly P). Granular poly P (volutin) can make up to 37% of the internal cell volume. This bacterium lacks the classic enzyme of poly P synthesis, class I polyphosphate kinase (PPK1), but it possesses two genes,
ppk2A
(corresponds to NCgl0880) and
ppk2B
(corresponds to NCgl2620), for putative class II (PPK2) PPKs. Deletion of
ppk2B
decreased PPK activity and cellular poly P content, while overexpression of
ppk2B
increased both PPK activity and cellular poly P content. Neither deletion nor overexpression of
ppk2A
changed specific activity of PPK or cellular poly P content significantly. Purified PPK2B of
C. glutamicum
is active as a homotetramer and formed poly P with an average chain length of about 125, as determined with
31
P nuclear magnetic resonance. The catalytic efficiency of
C. glutamicum
PPK2B was higher in the poly P-forming direction than for nucleoside triphosphate formation from poly P. The
ppk2B
deletion mutant, which accumulated very little poly P and grew as
C. glutamicum
wild type under phosphate-sufficient conditions, showed a growth defect under phosphate-limiting conditions.
Monitoring and Source Tracking of Tetracycline Resistance Genes in Lagoons and Groundwater Adjacent to Swine Production Facilities over a 3-Year Period
Authors: Koike S. , Krapac I. G. , Oliver H. D. , Yannarell A. C. , Chee-Sanford J. C. , Aminov R. I. , Mackie R. I. 
Published: June 2007

ABSTRACT

To monitor the dissemination of resistance genes into the environment, we determined the occurrence of tetracycline resistance (Tc
r
) genes in groundwater underlying two swine confinement operations. Monitoring well networks (16 wells at site A and 6 wells at site C) were established around the lagoons at each facility. Groundwater (
n
= 124) and lagoon (
n
= 12) samples were collected from the two sites at six sampling times from 2000 through 2003. Total DNA was extracted, and PCR was used to detect seven Tc
r
genes [
tet
(M),
tet
(O),
tet
(Q),
tet
(W),
tet
(C),
tet
(H), and
tet
(Z)]. The concentration of Tc
r
genes was quantified by real-time quantitative PCR. To confirm the Tc
r
gene source in groundwater, comparative analysis of
tet
(W) gene sequences was performed on groundwater and lagoon samples. All seven Tc
r
genes were continually detected in groundwater during the 3-year monitoring period at both sites. At site A, elevated detection frequency and concentration of Tc
r
genes were observed in the wells located down-gradient of the lagoon. Comparative analysis of
tet
(W) sequences revealed that the impacted groundwater contained gene sequences almost identical (99.8% identity) to those in the lagoon, but these genes were not found in background libraries. Novel sequence clusters and unique indigenous resistance gene pools were also found in the groundwater. Thus, antibiotic resistance genes in groundwater are affected by swine manure, but they are also part of the indigenous gene pool.
The First Agmatine/Cadaverine Aminopropyl Transferase: Biochemical and Structural Characterization of an Enzyme Involved in Polyamine Biosynthesis in the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Cacciapuoti Giovanna , Porcelli Marina , Moretti Maria Angela , Sorrentino Francesca , Concilio Luigi , Zappia Vincenzo , Liu Zhi-Jie , Tempel Wolfram , Schubot Florian , Rose John P. , Wang Bi-Cheng , Brereton Phillip S. , Jenney Francis E. , Adams Michael W. W. 
Published: June 2007

ABSTRACT

We report here the characterization of the first agmatine/cadaverine aminopropyl transferase (ACAPT), the enzyme responsible for polyamine biosynthesis from an archaeon. The gene PF0127 encoding ACAPT in the hyperthermophile
Pyrococcus furiosus
was cloned and expressed in
Escherichia coli
, and the recombinant protein was purified to homogeneity.
P. furiosus
ACAPT is a homodimer of 65 kDa. The broad substrate specificity of the enzyme toward the amine acceptors is unique, as agmatine, 1,3-diaminopropane, putrescine, cadaverine, and sym-nor-spermidine all serve as substrates. While maximal catalytic activity was observed with cadaverine, agmatine was the preferred substrate on the basis of the
k
cat
/

K
m

value.
P. furiosus
ACAPT is thermoactive and thermostable with an apparent melting temperature of 108°C that increases to 112°C in the presence of cadaverine. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. The crystal structure of the enzyme determined to 1.8-Å resolution confirmed its dimeric nature and provided insight into the proteolytic analyses as well as into mechanisms of thermal stability. Analysis of the polyamine content of
P. furiosus
showed that spermidine, cadaverine, and sym-nor-spermidine are the major components, with small amounts of sym-nor-spermine and
N
-(3-aminopropyl)cadaverine (APC). This is the first report in
Archaea
of an unusual polyamine APC that is proposed to play a role in stress adaptation.
Structure and Function of Cold Shock Proteins in Archaea
Authors: Giaquinto Laura , Curmi Paul M. G. , Siddiqui Khawar S. , Poljak Anne , DeLong Ed , DasSarma Shiladitya , Cavicchioli Ricardo 
Published: June 2007

ABSTRACT

Archaea are abundant and drive critical microbial processes in the Earth's cold biosphere. Despite this, not enough is known about the molecular mechanisms of cold adaptation and no biochemical studies have been performed on stenopsychrophilic archaea (e.g.,
Methanogenium frigidum
). This study examined the structural and functional properties of cold shock proteins (Csps) from archaea, including biochemical analysis of the Csp from
M. frigidum. csp
genes are present in most bacteria and some eucarya but absent from most archaeal genome sequences, most notably, those of all archaeal thermophiles and hyperthermophiles. In bacteria, Csps are small, nucleic acid binding proteins involved in a variety of cellular processes, such as transcription. In this study, archaeal Csp function was assessed by examining the ability of
csp
genes from psychrophilic and mesophilic
Euryarchaeota
and
Crenarchaeota
to complement a cold-sensitive growth defect in
Escherichia coli
. In addition, an archaeal gene with a cold shock domain (CSD) fold but little sequence identity to Csps was also examined. Genes encoding Csps or a CSD structural analog from three psychrophilic archaea rescued the
E. coli
growth defect. The three proteins were predicted to have a higher content of solvent-exposed basic residues than the noncomplementing proteins, and the basic residues were located on the nucleic acid binding surface, similar to their arrangement in
E. coli
CspA. The
M. frigidum
Csp was purified and found to be a single-domain protein that folds by a reversible two-state mechanism and to exhibit a low conformational stability typical of cold-adapted proteins. Moreover,
M. frigidum
Csp was characterized as binding
E. coli
single-stranded RNA, consistent with its ability to complement function in
E. coli
. The studies show that some Csp and CSD fold proteins have retained sufficient similarity throughout evolution in the
Archaea
to be able to function effectively in the
Bacteria
and that the function of the archaeal proteins relates to cold adaptation. The initial biochemical analysis of
M. frigidum
Csp has developed a platform for further characterization and demonstrates the potential for expanding molecular studies of proteins from this important archaeal stenopsychrophile.
Regulation of Carbon and Nitrogen Utilization by CbrAB and NtrBC Two-Component Systems in
Pseudomonas aeruginosa
Authors: Li Wei , Lu Chung-Dar 
Published: June 2007

ABSTRACT

The global effect of the CbrAB and NtrBC two-component systems on the control of carbon and nitrogen utilization in
Pseudomonas aeruginosa
was characterized by phenotype microarray analyses with single and double mutants and the isogenic parent strain. The tested compounds were clustered based on the growth phenotypes of these strains, and the results clearly demonstrated the pivotal roles of CbrAB and NtrBC in carbon and nitrogen utilization, respectively. Growth of the
cbrAB
deletion mutant on arginine, histidine, and polyamines used as the sole carbon source was abolished, while growth on the tricarboxylic acid (TCA) cycle intermediates was sustained. In this study, suppressors of the
cbr
mutant were selected from minimal medium containing
l
-arginine as the sole carbon and nitrogen source. These mutants fell into two groups according to the ability to utilize histidine. The genomic library of a histidine-positive suppressor mutant was constructed, and the corresponding suppressor gene was identified by complementation as an
ntrB
allele. Similar results were obtained from four additional suppressor mutants, and point mutations of these
ntrB
alleles resulting in the following changes in residues were identified, with implications for reduced phosphatase activities: L126W, D227A, P228L, and S229I. The Ntr systems of these
ntrB
mutants became constitutively active, as revealed by the activity profiles of glutamate dehydrogenase, glutamate synthase, and glutamine synthetase. As a result, these mutants not only regain the substrate-specific induction on catabolic arginine and histidine operons but are also expressed to higher levels than the wild type. While the Δ
cbrAB ntrB
(Con) mutant restored growth on many N-containing compounds used as the carbon sources, its capability to grow on TCA cycle intermediates and glucose was compromised when ammonium served as the sole nitrogen source, mostly due to an extreme imbalance of carbon and nitrogen regulatory systems. In summary, this study supports the notion that CbrAB and NtrBC form a network to control the C/N balance in
P. aeruginosa
. Possible molecular mechanisms of these two regulatory elements in the control of arginine and histidine operons used as the model systems are discussed.
Protein Modulator of Multidrug Efflux Gene Expression inPseudomonas aeruginosa
Authors: Daigle Denis M. , Cao Lily , Fraud Sebastien , Wilke Mark S. , Pacey Angela , Klinoski Rachael , Strynadka Natalie C. , Dean Charles R. , Poole Keith 
Published: June 2007

ABSTRACTnalCmultidrug-resistant mutants ofPseudomonas aeruginosashow enhanced expression of themexAB-oprMmultidrug efflux system as a direct result of the production of a ca. 6,100-Da protein, PA3719, in these mutants. Using a bacterial two-hybrid system, PA3719 was shown to interact in vivo with MexR, a repressor ofmexAB-oprMexpression. Isothermal titration calorimetry (ITC) studies confirmed a high-affinity interaction (equilibrium dissociation constant [KD], 158.0 ± 18.1 nM) of PA3719 with MexR in vitro. PA3719 binding to and formation of a complex with MexR obviated repressor binding to its operator, which overlaps the efflux operon promoter, suggesting thatmexAB-oprMhyperexpression innalCmutants results from PA3719 modulation of MexR repressor activity. Consistent with this, MexR repression ofmexAtranscription in an in vitro transcription assay was alleviated by PA3719. Mutations in MexR compromising its interaction with PA3719 in vivo were isolated and shown to be located internally and distributed throughout the protein, suggesting that they impacted PA3719 binding by altering MexR structure or conformation rather than by having residues interacting specifically with PA3719. Four of six mutant MexR proteins studied retained repressor activity even in analCstrain producing PA3719. Again, this is consistent with a PA3719 interaction with MexR being necessary to obviate MexR repressor activity. The gene encoding PA3719 has thus been renamedarmR(antirepressor forMexR). A representative “noninteracting” mutant MexR protein, MexRI104F, was purified, and ITC confirmed that it bound PA3719 with reduced affinity (5.4-fold reduced;KD, 853.2 ± 151.1 nM). Consistent with this, MexRI104Frepressor activity, as assessed using the in vitro transcription assay, was only weakly compromised by PA3719. Finally, two mutations (L36P and W45A) in ArmR compromising its interaction with MexR have been isolated and mapped to a putative C-terminal α-helix of the protein that alone is sufficient for interaction with MexR.
The Intracellular Concentration of Acetyl Phosphate in
Escherichia coli
Is Sufficient for Direct Phosphorylation of Two-Component Response Regulators
Authors: Klein Adam H. , Shulla Ana , Reimann Sylvia A. , Keating David H. , Wolfe Alan J. 
Published: June 2007

ABSTRACT

Acetyl phosphate, the intermediate of the AckA-Pta pathway, acts as a global signal in
Escherichia coli
. Although acetyl phosphate clearly signals through two-component response regulators, it remains unclear whether acetyl phosphate acts as a direct phospho donor or functions through an indirect mechanism. We used two-dimensional thin-layer chromatography to measure the relative concentrations of acetyl phosphate, acetyl coenzyme A, ATP, and GTP over the course of the entire growth curve. We estimated that the intracellular concentration of acetyl phosphate in wild-type cells reaches at least 3 mM, a concentration sufficient to activate two-component response regulators via direct phosphoryl transfer.
Role of Staphylococcal Phage and SaPI Integrase in Intra- and Interspecies SaPI Transfer
Authors: Maiques Elisa , Úbeda Carles , Tormo María Ángeles , Ferrer María Desamparados , Lasa Íñigo , Novick Richard P. , Penadés José R. 
Published: June 2007

ABSTRACT

SaPIbov2 is a member of the SaPI family of staphylococcal pathogenicity islands and is very closely related to SaPIbov1. Typically, certain temperate phages can induce excision and replication of one or more of these islands and can package them into special small phage-like particles commensurate with their genome sizes (referred to as the excision-replication-packaging [ERP] cycle). We have studied the phage-SaPI interaction in some depth using SaPIbov2, with special reference to the role of its integrase. We demonstrate here that SaPIbov2 can be induced to replicate by different staphylococcal phages. After replication, SaPIbov2 is efficiently encapsidated and transferred to recipient organisms, including different non-
Staphylococcus aureus
staphylococci, where it integrates at a SaPI-specific attachment site,

att
C

, by means of a self-coded integrase (Int). Phages that cannot induce the SaPIbov2 ERP cycle can transfer the island by
recA
-dependent classical generalized transduction and can also transfer it by a novel mechanism that requires the expression of SaPIbov2
int
in the recipient but not in the donor. It is suggested that this mechanism involves the encapsidation of standard transducing fragments containing the intact island followed by
int
-mediated excision, circularization, and integration in the recipient.
A comparative study of the quality of DNA obtained from fresh frozen and formalin-fixed decalcified paraffin-embedded bone marrow trephine biopsy specimens using two different methods
Authors: Talaulikar D , Gray J X , Shadbolt B , McNiven M , Dahlstrom J E 
Published: June 2007

Background:Given its prognostic value, there is renewed interest in molecular staging in non-Hodgkin’s lymphoma (NHL) using immunoglobulin heavy and light chain (IgH, IgL) gene rearrangements.Aims:To compare the efficiency of DNA amplification from fresh frozen and formalin-fixed decalcified paraffin-embedded (FFDPE) bone marrow trephines for use in molecular staging using two methods.Methods:After manually extracting DNA from 13 FFDPE and 14 fresh frozen trephine biopsy specimens, two methods were used to test for amplifiability: use of the amplification control master mix supplied in the In Vivo Scribe immunoglobulin heavy chain (IgH) clonality kit, which creates 5 amplicons between 96–600 base pairs (bp); and real-time amplification of the β-globin gene.Results:Using the first method, the mean maximum length of amplicons generated from FFDPE trephines was statistically lower at 300 bp compared to fresh frozen samples, all of which generated amplicons up to 600 bp in size (p<0.001). Real-time amplification of the β-globin gene showed that the mean crossing threshold of fresh frozen samples was statistically lower than that of FFDPE samples (23.48 (95% CI 22.47 to 24.48) vs 33.64 (95% CI 32.15 to 35.12); p<0.001).Conclusions:Although amplifiable DNA can be extracted from both fresh-frozen and FFDPE trephine samples for IgH/IgL analysis, freshly frozen specimens are superior as a source of template DNA, especially for higher base pair PCR products.