bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Homophenylalanine Biosynthetic Genes from the Cyanobacterium Nostoc punctiforme PCC73102 and Application to Its Microbial Production by Escherichia coli
Authors: Koketsu Kento , Mitsuhashi Satoshi , Tabata Kazuhiko 
Published: January 2013

ABSTRACT

l-Homophenylalanine (
l
-Hph) is a useful chiral building block for synthesis of several drugs, including angiotensin-converting enzyme inhibitors and the novel proteasome inhibitor carfilzomib. While the chemoenzymatic route of synthesis is fully developed, we investigated microbial production of
l
-Hph to explore the possibility of a more efficient and sustainable approach to
l
-Hph production. We hypothesized that
l
-Hph is synthesized from
l
-Phe via a mechanism homologous to 3-methyl-2-oxobutanoic acid conversion to 4-methyl-2-oxopentanoic acid during leucine biosynthesis. Based on bioinformatics analysis, we found three putative homophenylalanine biosynthesis genes,
hphA
(Npun_F2464),
hphB
(Npun_F2457), and
hphCD
(Npun_F2458), in the cyanobacterium
Nostoc punctiforme
PCC73102, located around the gene cluster responsible for anabaenopeptin biosynthesis. We constructed
Escherichia coli
strains harboring
hphABCD
-expressing plasmids and achieved the fermentative production of
l
-Hph from
l
-Phe. To our knowledge, this is the first identification of the genes responsible for homophenylalanine synthesis in any organism. Furthermore, to improve the low conversion efficiency of the initial strain, we optimized the expression of
hphA
,
hphB
, and
hphCD
, which increased the yield to ∼630 mg/liter. The
l
-Hph biosynthesis and
l
-Leu biosynthesis genes from
E. coli
were also compared. This analysis revealed that HphB has comparatively relaxed substrate specificity and can perform the function of LeuB, but HphA and HphCD show tight substrate specificity and cannot complement the LeuA and LeuC/LeuD functions, and vice versa. Finally, the range of substrate tolerance of the
l
-Hph-producing strain was examined, which showed that
m
-fluorophenylalanine,
o
-fluorophenylalanine, and
l
-tyrosine were accepted as substrates and that the corresponding homoamino acids were generated.
Direct Assessment of Metabolite Utilization by Pseudomonas aeruginosa during Growth on Artificial Sputum Medium
Authors: Behrends Volker , Geier Benedikt , Williams Huw D. , Bundy Jacob G. 
Published: January 2013

ABSTRACT

We grew
Pseudomonas aeruginosa
in LB and artificial sputum medium (ASM) (filtered and unfiltered) and quantified metabolite utilization and excretion by nuclear magnetic resonance (NMR) spectroscopy (metabolic footprinting or extracellular metabolomics). Utilization rates were similar between media, but there were differences in excretion—e.g., acetate was produced only in unfiltered ASM.
Stochasticity in Colonial Growth Dynamics of Individual Bacterial Cells
Authors: Koutsoumanis Konstantinos P. , Lianou Alexandra 
Published: January 2013

ABSTRACT

Conventional bacterial growth studies rely on large bacterial populations without considering the individual cells. Individual cells, however, can exhibit marked behavioral heterogeneity. Here, we present experimental observations on the colonial growth of 220 individual cells of
Salmonella enterica
serotype Typhimurium using time-lapse microscopy videos. We found a highly heterogeneous behavior. Some cells did not grow, showing filamentation or lysis before division. Cells that were able to grow and form microcolonies showed highly diverse growth dynamics. The quality of the videos allowed for counting the cells over time and estimating the kinetic parameters lag time (λ) and maximum specific growth rate (μ
max
) for each microcolony originating from a single cell. To interpret the observations, the variability of the kinetic parameters was characterized using appropriate probability distributions and introduced to a stochastic model that allows for taking into account heterogeneity using Monte Carlo simulation. The model provides stochastic growth curves demonstrating that growth of single cells or small microbial populations is a pool of events each one of which has its own probability to occur. Simulations of the model illustrated how the apparent variability in population growth gradually decreases with increasing initial population size (
N
0
). For bacterial populations with
N
0
of >100 cells, the variability is almost eliminated and the system seems to behave deterministically, even though the underlying law is stochastic. We also used the model to demonstrate the effect of the presence and extent of a nongrowing population fraction on the stochastic growth of bacterial populations.
Photosynthetic Bradyrhizobium sp. Strain ORS285 Is Capable of Forming Nitrogen-Fixing Root Nodules on Soybeans (Glycine max)
Authors: Giraud Eric , Xu Lei , Chaintreuil Clï¿½mence , Gargani Daniel , Gully Djamel , Sadowsky Michael J. 
Published: January 2013

ABSTRACT

The ability of photosynthetic
Bradyrhizobium
strains ORS285 and ORS278 to nodulate soybeans was investigated. While the
nod
gene-deficient ORS278 strain induced bumps only on soybean roots, the
nod
gene-containing ORS285 strain formed nitrogen-fixing nodules. However, symbiotic efficiencies differed drastically depending on both the soybean genotype used and the culture conditions tested.
A Hydrolase from Lactobacillus sakei Moonlights as a Transaminase
Authors: Sinz Quirin , Freiding Simone , Vogel Rudi F. , Schwab Wilfried 
Published: January 2013

ABSTRACT

Enzymatic transamination of amino acids yields α-keto acids and is the initial step for the production of volatile compounds that contribute to the sensory perception of fermented foods such as salami.
Lactobacillus sakei
is one of the lactic acid bacterial strains commonly used in starter cultures. Although the genome sequence of
L. sakei
23K lacks genes encoding typical branched-chain amino acid transaminases, transamination activity and the formation of amino acid-derived volatile metabolites could be demonstrated. A protein purified from
L. sakei
is held responsible for the transamination activity. By heterologous expression of the corresponding gene in
Escherichia coli
, we were able to characterize the transamination side activity of an enzyme annotated as a putative acylphosphatase (AcP). A transamination side activity of hen egg white lysozyme (HEWL) was also discovered. Both enzymes showed substrate specificity toward branched-chain and aromatic amino acids. AcP also accepted l-methionine. Activity was optimal at neutral pH for both enzymes, whereas AcP showed a significantly higher temperature optimum (55ï¿½C) than that of HEWL (37ï¿½C). Kinetic parameters revealed high affinity toward
l
-leucine for AcP (

K
m

= 1.85 mM) and toward
l
-isoleucine for HEWL (

K
m

= 3.79 mM). AcP seems to play a major role in the metabolism of amino acids in
L. sakei
.
High Frequency of Virulence Factor Genes
tdh
,
trh
, and
tlh
in Vibrio parahaemolyticus Strains Isolated from a Pristine Estuary
Authors: Gutierrez West Casandra K. , Klein Savannah L. , Lovell Charles R. 
Published: January 2013

ABSTRACT

Virulence factor genes encoding the thermostable direct hemolysin (
tdh
) and the thermostable direct hemolysin-related hemolysin (
trh
) are strongly correlated with virulence of the emergent human pathogen
Vibrio parahaemolyticus
. The gene encoding the thermolabile hemolysin (
tlh
) is also considered a signature molecular marker for the species. These genes are typically reported in very low percentages (1 to 2%) of nonclinical strains.
V. parahaemolyticus
strains were isolated from various niches within a pristine estuary (North Inlet, SC) and were screened for these genes using both newly designed PCR primers and more commonly used primers. DNA sequences of
tdh
and
trh
were recovered from 48% and 8.3%, respectively, of these North Inlet strains. The recovery of pathogenic
V. parahaemolyticus
strains in such high proportions from an estuarine ecosystem that is virtually free of anthropogenic influences indicates the potential for additional, perhaps environmental roles of the
tdh
and
trh
genes.
Glucose Triggers ATP Secretion from Bacteria in a Growth-Phase-Dependent Manner
Authors: Hironaka Ippei , Iwase Tadayuki , Sugimoto Shinya , Okuda Ken-ichi , Tajima Akiko , Yanaga Katsuhiko , Mizunoe Yoshimitsu 
Published: January 2013

ABSTRACT

ATP modulates immune cell functions, and ATP derived from gut commensal bacteria promotes the differentiation of T helper 17 (Th17) cells in the intestinal lamina propria. We recently reported that
Enterococcus gallinarum
, isolated from mice and humans, secretes ATP. We have since found and characterized several ATP-secreting bacteria. Of the tested enterococci,
Enterococcus mundtii
secreted the greatest amount of ATP (>2 μM/10
8
cells) after overnight culture. Glucose, not amino acids and vitamins, was essential for ATP secretion from
E. mundtii
. Analyses of energy-deprived cells demonstrated that glycolysis is the most important pathway for bacterial ATP secretion. Furthermore, exponential-phase
E. mundtii
and
Enterococcus faecalis
cells secrete ATP more efficiently than stationary-phase cells. Other bacteria, including
Pseudomonas aeruginosa
,
Escherichia coli
, and
Staphylococcus aureus
, also secrete ATP in exponential but not stationary phase. These results suggest that various gut bacteria, including commensals and pathogens, might secrete ATP at any growth phase and modulate immune cell function.
Thermostable Alcohol Dehydrogenase from Thermococcus kodakarensis KOD1 for Enantioselective Bioconversion of Aromatic Secondary Alcohols
Authors: Wu Xi , Zhang Chong , Orita Izumi , Imanaka Tadayuki , Fukui Toshiaki , Xing Xin-Hui 
Published: January 2013

ABSTRACT

A novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeon
Thermococcus kodakarensis
KOD1 (
Tk
ADH). The gene,
tk0845
, which encodes an aldo-keto reductase, was heterologously expressed in
Escherichia coli
. The enzyme was found to be a monomer with a molecular mass of 31 kDa. It was highly thermostable with an optimal temperature of 90ï¿½C and a half-life of 4.5 h at 95ï¿½C. The apparent

K
m

values for the cofactors NAD(P)
+
and NADPH were similar within a range of 66 to 127 μM.
Tk
ADH preferred secondary alcohols and accepted various ketones and aldehydes as substrates. Interestingly, the enzyme could oxidize 1-phenylethanol and its derivatives having substituents at the
meta
and
para
positions with high enantioselectivity, yielding the corresponding (
R
)-alcohols with optical purities of greater than 99.8% enantiomeric excess (ee).
Tk
ADH could also reduce 2,2,2-trifluoroacetophenone to (
R
)-2,2,2-trifluoro-1-phenylethanol with high enantioselectivity (>99.6% ee). Furthermore, the enzyme showed high resistance to organic solvents and was particularly highly active in the presence of H
2
O–20% 2-propanol and H
2
O–50%
n
-hexane or
n
-octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols.
Microfluidic Chip-Based Detection and Intraspecies Strain Discrimination of Salmonella Serovars Derived from Whole Blood of Septic Mice
Authors: Patterson Adriana S. , Heithoff Douglas M. , Ferguson Brian S. , Soh H. Tom , Mahan Michael J. , Plaxco Kevin W. 
Published: January 2013

ABSTRACTSalmonellais a zoonotic pathogen that poses a considerable public health and economic burden in the United States and worldwide. Resultant human diseases range from enterocolitis to bacteremia to sepsis and are acutely dependent on the particular serovar ofSalmonella entericasubsp.enterica, which comprises over 99% of human-pathogenicS. entericaisolates. Point-of-care methods for detection and strain discrimination ofSalmonellaserovars would thus have considerable benefit to medical, veterinary, and field applications that safeguard public health and reduce industry-associated losses. Here we describe a single, disposable microfluidic chip that supports isothermal amplification and sequence-specific detection and discrimination ofSalmonellaserovars derived from whole blood of septic mice. The integrated microfluidic electrochemical DNA (IMED) chip consists of an amplification chamber that supports loop-mediated isothermal amplification (LAMP), a rapid, single-temperature amplification method as an alternative to PCR that offers advantages in terms of sensitivity, reaction speed, and amplicon yield. The amplification chamber is connected via a microchannel to a detection chamber containing a reagentless, multiplexed (here biplex) sensing array for sequence-specific electrochemical DNA (E-DNA) detection of the LAMP products. Validation of the IMED device was assessed by the detection and discrimination ofS. entericasubsp.entericaserovars Typhimurium and Choleraesuis, the causative agents of enterocolitis and sepsis in humans, respectively. IMED chips conferred rapid (under 2 h) detection and discrimination of these strains at clinically relevant levels (<1,000 CFU/ml) from whole, unprocessed blood collected from septic animals. The IMED-based chip assay shows considerable promise as a rapid, inexpensive, and portable point-of-care diagnostic platform for the detection and strain-specific discrimination of microbial pathogens.
Frequent Occurrence of Extended-Spectrum Beta-Lactamase- and Transferable AmpC Beta-Lactamase-Producing Escherichia coli on Domestic Chicken Meat in Sweden
Authors: Bï¿½rjesson Stefan , Egervï¿½rn Maria , Lindblad Mats , Englund Stina 
Published: January 2013

ABSTRACT

Forty-four percent of Swedish chicken meat fillets were contaminated with extended-spectrum or transferable AmpC beta-lactamase-producing
Escherichia coli
strains. Isolates from Swedish chicken meat and broilers were closely related to isolates from chicken meat imported into Sweden; these results indicate a common source of the contamination.
Distinct and Redundant Roles of the Two MYST Histone Acetyltransferases Esa1 and Sas2 in Cell Growth and Morphogenesis of Candida albicans
Authors: Wang Xiongjun , Chang Peng , Ding Jianping , Chen Jiangye 
Published: January 2013

ABSTRACT

Candida albicans
is associated with humans, as both a harmless commensal organism and a pathogen. Adaption to human body temperature is extremely important for its growth and morphogenesis.
Saccharomyces cerevisiae
Esa1, a member of the MYST family HATs (histone acetyltransferases) and the catalytic subunit of the NuA4 complex, and its homologues in other eukaryotes have been shown to be essential for cell growth. To investigate the functional roles of two MYST family HATs, Esa1 and Sas2 in
C. albicans
, we deleted
ESA1
and
SAS2
in the
C. albicans
genome and performed cell growth analyses. Our results demonstrated that
C. albicans
Esa1 is not essential for general growth but is essential for filamentous growth. The
esa1/esa1
mutant cells exhibited sensitivity to thermal, genotoxic, and oxidative stresses but tolerance to cold, osmotic, and cell wall stresses. In contrast, the
sas2
/
sas2
mutant adapted to growth at higher temperatures and promoted filament formation at lower temperatures, resembling the phenotype of a
C. albicans
strain overexpressing
ESA1
. Cells with deletions of both
ESA1
and
SAS2
were inviable, reflecting the functional redundancy in cell growth.
C. albicans
Esa1 and Sas2 have distinct and synergistic effects on histone acetylation at H4K5, H4K12, and H4K16. Esa1 contributes mainly to acetylation of H4K5 and H4K12, whereas Sas2 contributes to acetylation of H4K16. Our findings suggest that
C. albicans
Esa1 and Sas2 play opposite roles in cell growth and morphogenesis and contribute coordinately to histone acetylation and gene regulation.
Effects of a Defective Endoplasmic Reticulum-Associated Degradation Pathway on the Stress Response, Virulence, and Antifungal Drug Susceptibility of the Mold Pathogen Aspergillus fumigatus
Authors: Krishnan Karthik , Feng Xizhi , Powers-Fletcher Margaret V. , Bick Gregory , Richie Daryl L. , Woollett Laura A. , Askew David S. 
Published: January 2013

ABSTRACT

Proteins that are destined for release outside the eukaryotic cell, insertion into the plasma membrane, or delivery to intracellular organelles are processed and folded in the endoplasmic reticulum (ER). An imbalance between the level of nascent proteins entering the ER and the organelle's ability to manage that load results in the accumulation of unfolded proteins. Terminally unfolded proteins are disposed of by ER-associated degradation (ERAD), a pathway that transports the aberrant proteins across the ER membrane into the cytosol for proteasomal degradation. The ERAD pathway was targeted in the mold pathogen
Aspergillus fumigatus
by deleting the
hrdA
gene, encoding the
A. fumigatus
ortholog of Hrd1, the E3 ubiquitin ligase previously shown to contribute to ERAD in other species. Loss of HrdA was associated with impaired degradation of a folding-defective ERAD substrate, CPY*, as well as activation of the unfolded-protein response (UPR). The Δ
hrdA
mutant showed resistance to voriconazole and reduced thermotolerance but was otherwise unaffected by a variety of environmental stressors. A double-deletion mutant deficient in both HrdA and another component of the same ERAD complex, DerA, was defective in secretion and showed hypersensitivity to ER, thermal, and cell wall stress. However, the Δ
hrdA
Δ
derA
mutant remained virulent in mouse and insect infection models. These data demonstrate that HrdA and DerA support complementary ERAD functions that promote survival under conditions of ER stress but are dispensable for virulence in the host environment.
Reduction of Ribosome Level Triggers Flocculation of Fission Yeast Cells
Authors: Li Rongpeng , Li Xuesong , Sun Lei , Chen Feifei , Liu Zhenxing , Gu Yuyu , Gong Xiaoyan , Liu Zhonghua , Wei Hua , Huang Ying , Yuan Sheng 
Published: January 2013

ABSTRACT

Deletion of ribosomal protein L32 genes resulted in a nonsexual flocculation of fission yeast. Nonsexual flocculation also occurred when two other ribosomal protein genes,
rpl21-2
and
rpl9-2
, were deleted. However, deletion of two nonribosomal protein genes,
mpg
and
fbp
, did not cause flocculation. Overall transcript levels of
rpl32
in
rpl32-1
Δ and
rpl32-2
Δ cells were reduced by 35.9% and 46.9%, respectively, and overall ribosome levels in
rpl32-1
Δ and
rpl32-2
Δ cells dropped 31.1% and 27.8%, respectively, compared to wild-type cells. Interestingly, ribosome protein expression levels and ribosome levels were also reduced greatly in sexually flocculating diploid YHL6381/WT (
h
+
/h
−
) cells compared to a mixture of YHL6381 (
h
+
) and WT (
h
−
) nonflocculating haploid cells. Transcriptome analysis indicated that the reduction of ribosomal levels in sexual flocculating cells was caused by more-extensive suppression of ribosomal biosynthesis gene expression, while the reduction of ribosomal levels caused by deleting ribosomal protein genes in nonsexual flocculating cells was due to an imbalance between ribosomal proteins. We propose that once the reduction of ribosomal levels is below a certain threshold value, flocculation is triggered.
Conserved Gene Regulatory Function of the Carboxy-Terminal Domain of Dictyostelid C-Module-Binding Factor
Authors: Schmith Anika , Groth Marco , Ratka Josephine , Gatz Sara , Spaller Thomas , Siol Oliver , Glöckner Gernot , Winckler Thomas 
Published: January 2013

ABSTRACT

C-module-binding factor A (CbfA) is a jumonji-type transcription regulator that is important for maintaining the expression and mobility of the retrotransposable element TRE5-A in the social amoeba
Dictyostelium discoideum
. CbfA-deficient cells have lost TRE5-A retrotransposition, are impaired in the ability to feed on bacteria, and do not enter multicellular development because of a block in cell aggregation. In this study, we performed Illumina RNA-seq of growing CbfA mutant cells to obtain a list of CbfA-regulated genes. We demonstrate that the carboxy-terminal domain of CbfA alone is sufficient to mediate most CbfA-dependent gene expression. The carboxy-terminal domain of CbfA from the distantly related social amoeba
Polysphondylium pallidum
restored the expression of CbfA-dependent genes in the
D. discoideum
CbfA mutant, indicating a deep conservation in the gene regulatory function of this domain in the dictyostelid clade. The CbfA-like protein CbfB displays ∼25% sequence identity with CbfA in the amino-terminal region, which contains a JmjC domain and two zinc finger regions and is thought to mediate chromatin-remodeling activity. In contrast to CbfA proteins, where the carboxy-terminal domains are strictly conserved in all dictyostelids, CbfB proteins have completely unrelated carboxy-terminal domains. Outside the dictyostelid clade, CbfA-like proteins with the CbfA-archetypical JmjC/zinc finger arrangement and individual carboxy-terminal domains are prominent in filamentous fungi but are not found in yeasts, plants, and metazoans. Our data suggest that two functional regions of the CbfA-like proteins evolved at different rates to allow the occurrence of species-specific adaptation processes during genome evolution.
Two Roles for Aconitase in the Regulation of Tricarboxylic Acid Branch Gene Expression in Bacillus subtilis
Authors: Pechter Kieran B. , Meyer Frederik M. , Serio Alisa W. , Stülke Jörg , Sonenshein Abraham L. 
Published: January 2013

ABSTRACT

Previously, it was shown that an aconitase (
citB
) null mutation results in a vast overaccumulation of citrate in the culture fluid of growing
Bacillus subtilis
cells, a phenotype that causes secondary effects, including the hyperexpression of the
citB
promoter.
B. subtilis
aconitase is a bifunctional protein; to determine if either or both activities of aconitase were responsible for this phenotype, two strains producing different mutant forms of aconitase were constructed, one designed to be enzymatically inactive (C450S [
citB2
]) and the other designed to be defective in RNA binding (R741E [
citB7
]). The
citB2
mutant was a glutamate auxotroph and accumulated citrate, while the
citB7
mutant was a glutamate prototroph. Unexpectedly, the
citB7
strain also accumulated citrate. Both mutant strains exhibited overexpression of the
citB
promoter and accumulated high levels of aconitase protein. These strains and the
citB
null mutant also exhibited increased levels of citrate synthase protein and enzyme activity in cell extracts, and the major citrate synthase (
citZ
) transcript was present at higher-than-normal levels in the
citB
null mutant, due at least in part to a >3-fold increase in the stability of the
citZ
transcript compared to the wild type. Purified
B. subtilis
aconitase bound to the
citZ
5′ leader RNA
in vitro
, but the mutant proteins did not. Together, these data suggest that wild-type aconitase binds to and destabilizes the
citZ
transcript in order to maintain proper cell homeostasis by preventing the overaccumulation of citrate.