bluuebunny/crossref_metadata_2025_split huggingface dataset:
Malic Acid Production by
Saccharomyces cerevisiae
: Engineering of Pyruvate Carboxylation, Oxaloacetate Reduction, and Malate Export
Authors: Zelle Rintze M. , de Hulster Erik , van Winden Wouter A. , de Waard Pieter , Dijkema Cor , Winkler Aaron A. , Geertman Jan-Maarten A. , van Dijken Johannes P. , Pronk Jack T. , van Maris Antonius J. A. 
Published: March 2008

ABSTRACT

Malic acid is a potential biomass-derivable “building block” for chemical synthesis. Since wild-type
Saccharomyces cerevisiae
strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO
2
-fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose)
−1
. A previously engineered glucose-tolerant, C
2
-independent pyruvate decarboxylase-negative
S. cerevisiae
strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by
PYC2
, (ii) high-level expression of an allele of the
MDH3
gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the
Schizosaccharomyces pombe
malate transporter gene Sp
MAE1
. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter
−1
at a malate yield of 0.42 mol (mol glucose)
−1
. Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on
13
C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved.
High-Level Folate Production in Fermented Foods by the B
12
Producer
Lactobacillus reuteri
JCM1112
Authors: Santos Filipe , Wegkamp Arno , de Vos Willem M. , Smid Eddy J. , Hugenholtz Jeroen 
Published: March 2008

ABSTRACT

We observed that
Lactobacillus reuteri
JCM1112 produces B
12
and folate. However, the folate/B
12
mass ratio found was far below that desired for human consumption (∼170:1). We used metabolic engineering applying genetic and physiological approaches to improve this ratio and developed a generic and natural process that significantly increases folate production.
Biogeographic and Phylogenetic Diversity of Thermoacidophilic Cyanidiales in Yellowstone National Park, Japan, and New Zealand
Authors: Toplin J. A. , Norris T. B. , Lehr C. R. , McDermott T. R. , Castenholz R. W. 
Published: March 2008

ABSTRACT

Members of the rhodophytan order Cyanidiales are unique among phototrophs in their ability to live in extreme environments that combine low pH levels (∼0.2 to 4.0) and moderately high temperatures of 40 to 56°C. These unicellular algae occur in far-flung volcanic areas throughout the earth. Three genera (
Cyanidium
,
Galdieria
, and
Cyanidioschyzon
) are recognized. The phylogenetic diversity of culture isolates of the Cyanidiales from habitats throughout Yellowstone National Park (YNP), three areas in Japan, and seven regions in New Zealand was examined by using the chloroplast RuBisCO large subunit gene (
rbcL
) and the 18S rRNA gene. Based on the nucleotide sequences of both genes, the YNP isolates fall into two groups, one with high identity to
Galdieria sulphuraria
(type II) and another that is by far the most common and extensively distributed Yellowstone type (type IA). The latter is a spherical, walled cell that reproduces by internal divisions, with a subsequent release of smaller daughter cells. This type, nevertheless, shows a 99 to 100% identity to
Cyanidioschyzon merolae
(type IB), which lacks a wall, divides by “fission”-like cytokinesis into two daughter cells, and has less than 5% of the cell volume of type IA. The evolutionary and taxonomic ramifications of this disparity are discussed. Although the 18S rRNA and
rbcL
genes did not reveal diversity among the numerous isolates of type IA, chloroplast short sequence repeats did show some variation by location within YNP. In contrast, Japanese and New Zealand strains showed considerable diversity when we examined only the sequences of 18S and
rbcL
genes. Most exhibited identities closer to
Galdieria maxima
than to other strains, but these identities were commonly as low as 91 to 93%. Some of these Japanese and New Zealand strains probably represent undescribed species that diverged after long-term geographic isolation.
Application of Functional Genomics to Pathway Optimization for Increased Isoprenoid Production
Authors: Kizer Lance , Pitera Douglas J. , Pfleger Brian F. , Keasling Jay D. 
Published: March 2008

ABSTRACT

Producing complex chemicals using synthetic metabolic pathways in microbial hosts can have many advantages over chemical synthesis but is often complicated by deleterious interactions between pathway intermediates and the host cell metabolism. With the maturation of functional genomic analysis, it is now technically feasible to identify modes of toxicity associated with the accumulation of foreign molecules in the engineered bacterium. Previously,
Escherichia coli
was engineered to produce large quantities of isoprenoids by creating a mevalonate-based isopentenyl pyrophosphate biosynthetic pathway (V. J. J. Martin et al., Nat. Biotechnol.
21:
796-802, 2003). The engineered
E. coli
strain produced high levels of isoprenoids, but further optimization led to an imbalance in carbon flux and the accumulation of the pathway intermediate 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA), which proved to be cytotoxic to
E. coli
. Using both DNA microarray analysis and targeted metabolite profiling, we have studied
E. coli
strains inhibited by the intracellular accumulation of HMG-CoA. Our results indicate that HMG-CoA inhibits fatty acid biosynthesis in the microbial host, leading to generalized membrane stress. The cytotoxic effects of HMG-CoA accumulation can be counteracted by the addition of palmitic acid (16:0) and, to a lesser extent, oleic acid (
cis
-Δ
9
-18:1) in the growth medium. This work demonstrates the utility of using transcriptomic and metabolomic methods to optimize synthetic biological systems.
Imaging of Long-Distance α-Aminoisobutyric Acid Translocation Dynamics during Resource Capture by
Serpula lacrymans
Authors: Tlalka Monika , Fricker Mark , Watkinson Sarah 
Published: March 2008

ABSTRACT

α-Aminoisobutyric acid (AIB) is a nonmetabolized amino acid analogue of alanine, which at low (μM) concentrations acts as a tracer for amino acid movements. At high concentrations (mM), it competitively inhibits membrane transport and metabolism of protein amino acids and acts as a systemic translocated inhibitor of mycelial extension in fungi. AIB can control mycelial spread of the basidiomycete
Serpula lacrymans
, the cause of brown rot of wood in buildings. However, it is not known how effectively the inhibitor is distributed throughout the mycelium. Realistically heterogeneous microcosms, in which the fungus grew across nutritionally inert sand to colonize discrete wood resources, were used to investigate patterns of inhibition and translocation following local application of AIB. At a 0.1 M concentration, locally applied AIB caused immediate arrest of extension throughout the whole mycelium, maintained for a 6-week experimental period. The dynamics of translocation of subtoxic amounts of [1-
14
C]AIB ([
14
C]AIB) were mapped by photon-counting scintillation imaging in conjunction with destructive harvest to establish the velocity, direction, and rate of translocation and the extent of [
14
C]AIB reallocation accompanying the invasion of fresh wood. Locally applied [
14
C]AIB was distributed throughout complex mycelial networks within 2 h of application, becoming localized in growing margins by 12 h. Encounter with a fresh wood resource triggered a widespread response, causing withdrawal of [
14
C]AIB from throughout the network, accompanied by accumulation in the newly colonized wood and associated mycelium. The results are discussed in the context of nutrient dynamics in wood decomposer fungi and the mechanism of the amino acid reallocation response.
Efficient Production of
l
-Ribose with a Recombinant
Escherichia coli
Biocatalyst
Authors: Woodyer Ryan D. , Wymer Nathan J. , Racine F. Michael , Khan Shama N. , Saha Badal C. 
Published: March 2008

ABSTRACT

A new synthetic platform with potential for the production of several rare sugars, with
l
-ribose as the model target, is described. The gene encoding the unique NAD-dependent mannitol-
1
-dehydrogenase (MDH) from
Apium graveolens
(garden celery) was synthetically constructed for optimal expression in
Escherichia coli
. This MDH enzyme catalyzes the interconversion of several polyols and their
l
-sugar counterparts, including the conversion of ribitol to
l
-ribose. Expression of recombinant MDH in the active form was successfully achieved, and one-step purification was demonstrated. Using the created recombinant
E. coli
strain as a whole-cell catalyst, the synthetic utility was demonstrated for production of
l
-ribose, and the system was improved using shaken flask experiments. It was determined that addition of 50 to 500 μM ZnCl
2
and addition of 5 g/liter glycerol both improved production. The final levels of conversion achieved were >70% at a concentration of 40 g/liter and >50% at a concentration of 100 g/liter. The best conditions determined were then scaled up to a 1-liter fermentation that resulted in 55% conversion of 100 g/liter ribitol in 72 h, for a volumetric productivity of 17.4 g liter
−1
day
−1
. This system represents a significantly improved method for the large-scale production of
l
-ribose.
Novel Root Fungal Consortium Associated with a Dominant Desert Grass
Authors: Porras-Alfaro Andrea , Herrera Jose , Sinsabaugh Robert L. , Odenbach Kylea J. , Lowrey Timothy , Natvig Donald O. 
Published: March 2008

ABSTRACT

The broad distribution and high colonization rates of plant roots by a variety of endophytic fungi suggest that these symbionts have an important role in the function of ecosystems. Semiarid and arid lands cover more than one-third of the terrestrial ecosystems on Earth. However, a limited number of studies have been conducted to characterize root-associated fungal communities in semiarid grasslands. We conducted a study of the fungal community associated with the roots of a dominant grass,
Bouteloua gracilis
, at the Sevilleta National Wildlife Refuge in New Mexico. Internal transcribed spacer ribosomal DNA sequences from roots collected in May 2005, October 2005, and January 2006 were amplified using fungal-specific primers, and a total of 630 sequences were obtained, 69% of which were novel (less than 97% similarity with respect to sequences in the NCBI database).
B. gracilis
roots were colonized by at least 10 different orders, including endophytic, coprophilous, mycorrhizal, saprophytic, and plant pathogenic fungi. A total of 51 operational taxonomic units (OTUs) were found, and diversity estimators did not show saturation. Despite the high diversity found within
B. gracilis
roots, the root-associated fungal community is dominated by a novel group of dark septate fungi (DSF) within the order Pleosporales. Microscopic analysis confirmed that
B. gracilis
roots are highly colonized by DSF. Other common orders colonizing the roots included Sordariales, Xylariales, and Agaricales. By contributing to drought tolerance and nutrient acquisition, DSF may be integral to the function of arid ecosystems.
Archaea
Dominate the Ammonia-Oxidizing Community in the Rhizosphere of the Freshwater Macrophyte
Littorella uniflora
Authors: Herrmann Martina , Saunders Aaron M. , Schramm Andreas 
Published: March 2008

ABSTRACT

Archaeal and bacterial ammonia monooxygenase genes (
amoA
) had similar low relative abundances in freshwater sediment. In the rhizosphere of the submersed macrophyte
Littorella uniflora
, archaeal
amoA
was 500- to >8,000-fold enriched compared to bacterial
amoA
, suggesting that the enhanced nitrification activity observed in the rhizosphere was due to ammonia-oxidizing
Archaea
.
The R1 Conjugative Plasmid Increases
Escherichia coli
Biofilm Formation through an Envelope Stress Response
Authors: Yang Xiaole , Ma Qun , Wood Thomas K. 
Published: March 2008

ABSTRACT

Differential gene expression in biofilm cells suggests that adding the derepressed conjugative plasmid R1
drd
19 increases biofilm formation by affecting genes related to envelope stress (
rseA
and
cpxAR
), biofilm formation (
bssR
and
cstA
), energy production (
glpDFK
), acid resistance (
gadABCEX
and
hdeABD
), and cell motility (
csgBEFG, yehCD, yadC
, and
yfcV
); genes encoding outer membrane proteins (
ompACF
), phage shock proteins (
pspABCDE
), and cold shock proteins (
cspACDEG
); and phage-related genes. To investigate the link between the identified genes and biofilm formation upon the addition of R1
drd
19, 40 isogenic mutants were classified according to their different biofilm formation phenotypes. Cells with class I mutations (those in
rseA, bssR, cpxA
, and
ompA
) exhibited no difference from the wild-type strain in biofilm formation and no increase in biofilm formation upon the addition of R1
drd
19. Cells with class II mutations (those in
gatC, yagI, ompC, cspA, pspD, pspB, ymgB, gadC, pspC, ymgA, slp, cpxP, cpxR, cstA, rseC, ompF
, and
yqjD
) displayed increased biofilm formation compared to the wild-type strain but decreased biofilm formation upon the addition of R1
drd
19. Class III mutants showed increased biofilm formation compared to the wild-type strain and increased biofilm formation upon the addition of R1
drd
19. Cells with class IV mutations displayed increased biofilm formation compared to the wild-type strain but little difference upon the addition of R1
drd
19, and class V mutants exhibited no difference from the wild-type strain but increased biofilm formation upon the addition of R1
drd
19. Therefore, proteins encoded by the genes corresponding to the class I mutant phenotype are involved in R1
drd
19-promoted biofilm formation, primarily through their impact on cell motility. We hypothesize that the pili formed upon the addition of the conjugative plasmid disrupt the membrane (induce
ompA
) and activate the two-component system CpxAR as well as the other envelope stress response system, RseA-σ
E
, both of which, along with BssR, play a key role in bacterial biofilm formation.
Impact of Vacuum Cooling onEscherichia coliO157:H7 Infiltration into Lettuce Tissue
Authors: Li Haiping , Tajkarimi Mehrdad , Osburn Bennie I. 
Published: March 2008

ABSTRACTVacuum cooling is a common practice in the California leafy green industry. This study addressed the impact of vacuum cooling on the infiltration ofEscherichia coliO157:H7 into lettuce as part of the risk assessment responding to theE. coliO157:H7 outbreaks associated with leafy green produce from California. Vacuum cooling significantly increased the infiltration ofE. coliO157:H7 into the lettuce tissue (2.65E+06 CFU/g) compared to the nonvacuumed condition (1.98E+05 CFU/g). A stringent surface sterilization and quadruple washing could not eliminate the internalized bacteria from lettuce. It appeared that vacuuming forcibly changed the structure of lettuce tissue such as the stomata, suggesting a possible mechanism ofE. coliO157:H7 internalization. Vacuuming also caused a lower reduction rate ofE. coliO157:H7 in stored lettuce leaves than that for the nonvacuumed condition.
Randomly Amplified Polymorphic DNA PCR as a Tool for Assessment of Marine Viral Richness
Authors: Winget Danielle M. , Wommack K. Eric 
Published: March 2008

ABSTRACT
Recent discoveries have uncovered considerable genetic diversity among aquatic viruses and raised questions about the variability of this diversity within and between environments. Studies of the temporal and spatial dynamics of aquatic viral assemblages have been hindered by the lack of a common genetic marker among viruses for rapid diversity assessments. Randomly amplified polymorphic DNA (RAPD) PCR bypasses this obstacle by sampling at the genetic level without requiring viral isolation or previous sequence knowledge. In this study, the utility of RAPD-PCR for assessing DNA viral richness within Chesapeake Bay water samples was evaluated. RAPD-PCR using single 10-mer oligonucleotide primers successfully produced amplicons from a variety of viral samples, and banding patterns were highly reproducible, indicating that each band likely represents a single amplicon originating from viral template DNA. In agreement with observations from other community profiling techniques, resulting RAPD-PCR banding patterns revealed more temporal than spatial variability in Chesapeake Bay virioplankton assemblages. High-quality hybridization probes and sequence information were also easily generated from single RAPD-PCR products or whole reactions. Thus, the RAPD-PCR technique appears to be practical and efficient for routine use in high-resolution viral diversity studies by providing assemblage comparisons through fingerprinting, probing, or sequence information.
Isolation and Identification of Rhizoxin Analogs from
Pseudomonas fluorescens
Pf-5 by Using a Genomic Mining Strategy
Authors: Loper Joyce E. , Henkels Marcella D. , Shaffer Brenda T. , Valeriote Frederick A. , Gross Harald 
Published: March 2008

ABSTRACT

The products synthesized from a hybrid polyketide synthase/nonribosomal peptide synthetase gene cluster in the genome of
Pseudomonas fluorescens
Pf-5 were identified using a genomics-guided strategy involving insertional mutagenesis and subsequent metabolite profiling. Five analogs of rhizoxin, a 16-member macrolide with antifungal, phytotoxic, and antitumor activities, were produced by Pf-5, but not by a mutant with an insertion in the gene cluster. The five rhizoxin analogs, one of which had not been described previously, were differentially toxic to two agriculturally important plant pathogens,
Botrytis cinerea
and
Phytophthora ramorum
. The rhizoxin analogs also caused swelling of rice roots, a symptom characteristic of rhizoxin itself, but were less toxic to pea and cucumber roots. Of the rhizoxin analogs produced by Pf-5, the predominant compound, WF-1360 F, and the newly described compound 22
Z
-WF-1360 F were most toxic against the two plant pathogens and three plant species. These rhizoxin analogs were tested against a panel of human cancer lines, and they exhibited potent but nonselective cytotoxicity. This study highlights the value of the genomic sequence of the soil bacterium
P. fluorescens
Pf-5 in providing leads for the discovery of novel metabolites with significant biological properties.
Identification of Differentially Regulated
Francisella tularensis
Genes by Use of a Newly Developed Tn
5
-Based Transposon Delivery System
Authors: Buchan Blake W. , McLendon Molly K. , Jones Bradley D. 
Published: March 2008

ABSTRACT

Francisella tularensis
is the etiologic agent of an intracellular systemic infection of the lymphatic system in humans called tularemia. The organism has become the subject of considerable research interest due to its classification as a category A select agent by the CDC. To aid genetic analysis of this pathogen, we have constructed a temperature-sensitive Tn
5
-based transposon delivery system that is capable of generating chromosomal reporter fusions with
lacZ
or
luxCDABE
, enabling us to monitor gene expression. Transposition is catalyzed by the hyperactive Tn
5
transposase, whose expression is driven by the
Francisella groES
promoter. When high-temperature selection (42°C) is applied to a bacterial culture carrying the transposon delivery plasmid, ∼0.1% of the population is recovered with Tn
5
insertions in the chromosome. Nucleotide sequence analysis of a sample of mutants revealed that the insertions occur randomly throughout the chromosome. The kanamycin-selectable marker of the transposon is also flanked by FLP recombination target sequences that allow deletion of the antibiotic resistance gene when desired. This system has been used to generate transposon mutant libraries for the
F. tularensis
live vaccine strain as well as two different virulent
F. tularensis
strains. Chromosomal reporters delivered with the transposon were used to identify genes upregulated by growth in Chamberlain's defined medium. Genes in the
fsl
operon, reported to be involved in iron acquisition, as well as genes in the
igl
gene cluster were among those identified by the screen. Further experiments implicate the ferric uptake regulator (Fur) protein in the negative regulation of
fsl
but not
igl
reporters, which occurs in an iron-dependent manner. Our results indicate that we have created a valuable new transposon that can be used to identify and characterize virulence genes in
F. tularensis
strains.
MADS-Box Transcription Factor Mig1 Is Required for Infectious Growth in
Magnaporthe grisea
Authors: Mehrabi Rahim , Ding Shengli , Xu Jin-Rong 
Published: March 2008

ABSTRACT

Magnaporthe grisea
is a model fungus for studying fungus-plant interactions. Two mitogen-activated protein (MAP) kinase genes,
PMK1
and
MPS1
, have been implicated in regulating plant infection processes in
M. grisea
. However, transcription factors activated by these MAP kinases are not well studied. In this study we functionally characterized the
MIG1
gene that encodes a MADS-box transcription factor homologous to
Saccharomyces cerevisiae
Rlm1. In yeast two-hybrid assays,
MIG1
interacts with
MPS1
, suggesting that
MIG1
may function downstream from the
MPS1
pathway. The
mig1
deletion mutant had a normal growth rate and formed melanized appressoria, but it was nonpathogenic and failed to infect rice leaves through wounds. Appressoria formed by the
mig1
mutant developed penetration pegs and primary infectious hyphae, but further differentiation of the secondary infectious hyphae inside live plant cells was blocked. However, the
mig1
mutant formed infectious hypha-like structures in heat-killed plant cells or cellophane membranes. In transformants expressing the
MIG1
-
GFP
fusion, green fluorescent protein (GFP) signals were not detectable in vegetative hyphae and conidiophores. Mig1-GFP was localized to nuclei in conidia, appressoria, and infectious hyphae. Deletion of the MADS box had no effect on the expression and localization of the
MIG1-GFP
fusion but eliminated its ability to complement the
mig1
mutant. These results suggest that
MIG1
may be required for overcoming plant defense responses and the differentiation of secondary infectious hyphae in live plant cells. The MADS-box domain is essential for the function of
MIG1
but dispensable for its nuclear localization, which may be associated with the activation of
MIG1
by
MPS1
during conidiation and plant infection.