bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning and Characterization of a Novel Esterase from Rhodococcus sp. for Highly Enantioselective Synthesis of a Chiral Cilastatin Precursor
Authors: Zhang Yan , Pan Jiang , Luan Zheng-Jiao , Xu Guo-Chao , Park Sunghoon , Xu Jian-He 
Published: September 2014

ABSTRACT

A novel nonheme chloroperoxidase (
Rh
Est1), with promiscuous esterase activity for enantioselective hydrolysis of ethyl (
S
)-2,2-dimethylcyclopropanecarboxylate, was identified from a shotgun library of
Rhodococcus
sp. strain ECU1013.
Rh
Est1 was overexpressed in
Escherichia coli
BL21(DE3), purified to homogeneity, and functionally characterized. Fingerprinting analysis revealed that
Rh
Est1 prefers
para
-nitrophenyl (
p
NP) esters of short-chain acyl groups.
p
NP esters with a cyclic acyl moiety, especially that with a cyclobutanyl group, were also substrates for
Rh
Est1. The

K
m

values for methyl 2,2-dimethylcyclopropanecarboxylate (DmCpCm) and ethyl 2,2-dimethylcyclopropane carboxylate (DmCpCe) were 0.25 and 0.43 mM, respectively.
Rh
Est1 could serve as an efficient hydrolase for the bioproduction of optically pure (
S
)-2,2-dimethyl cyclopropane carboxylic acid (DmCpCa), which is an important chiral building block for cilastatin. As much as 0.5 M DmCpCe was enantioselectively hydrolyzed into (
S
)-DmCpCa, with a molar yield of 47.8% and an enantiomeric excess (
ee
) of 97.5%, indicating an extremely high enantioselectivity (
E
= 240) of this novel and unique biocatalyst for green manufacturing of highly valuable chiral chemicals.
Time-Scaled Evolutionary Analysis of the Transmission and Antibiotic Resistance Dynamics of Staphylococcus aureus Clonal Complex 398
Authors: Ward M. J. , Gibbons C. L. , McAdam P. R. , van Bunnik B. A. D. , Girvan E. K. , Edwards G. F. , Fitzgerald J. R. , Woolhouse M. E. J. 
Published: September 2014

ABSTRACT

Staphylococcus aureus
clonal complex 398 (CC398) is associated with disease in humans and livestock, and its origins and transmission have generated considerable interest. We performed a time-scaled phylogenetic analysis of CC398, including sequenced isolates from the United Kingdom (Scotland), along with publicly available genomes. Using state-of-the-art methods for mapping traits onto phylogenies, we quantified transitions between host species to identify sink and source populations for CC398 and employed a novel approach to investigate the gain and loss of antibiotic resistance in CC398 over time. We identified distinct human- and livestock-associated CC398 clades and observed multiple transmissions of CC398 from livestock to humans and between countries, lending quantitative support to previous reports. Of note, we identified a subclade within the livestock-associated clade comprised of isolates from hospital environments and newborn babies, suggesting that livestock-associated CC398 is capable of onward transmission in hospitals. In addition, our analysis revealed significant differences in the dynamics of resistance to methicillin and tetracycline related to contrasting historical patterns of antibiotic usage between the livestock industry and human medicine. We also identified significant differences in patterns of gain and loss of different tetracycline resistance determinants, which we ascribe to epistatic interactions between the resistance genes and/or differences in the modes of inheritance of the resistance determinants.
Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System
Authors: Bradshaw Marite , Tepp William H. , Whitemarsh Regina C. M. , Pellett Sabine , Johnson Eric A. 
Published: September 2014

ABSTRACT

Clostridium botulinum
subtype A4 neurotoxin (BoNT/A4) is naturally expressed in the dual-toxin-producing
C. botulinum
strain 657Ba at 100× lower titers than BoNT/B. In this study, we describe purification of recombinant BoNT/A4 (rBoNT/A4) expressed in a nonsporulating and nontoxigenic
C. botulinum
expression host strain. The rBoNT/A4 copurified with nontoxic toxin complex components provided in
trans
by the expression host and was proteolytically cleaved to the active dichain form. Activity of the recombinant BoNT/A4 in mice and in human neuronal cells was about 1,000-fold lower than that of BoNT/A1, and the recombinant BoNT/A4 was effectively neutralized by botulism heptavalent antitoxin. A previous report using recombinant truncated BoNT/A4 light chain (LC) expressed in
Escherichia coli
has indicated reduced stability and activity of BoNT/A4 LC compared to BoNT/A1 LC, which was surmounted by introduction of a single-amino-acid substitution, I264R. In order to determine whether this mutation would also affect the holotoxin activity of BoNT/A4, a recombinant full-length BoNT/A4 carrying this mutation as well as a second mutation predicted to increase solubility (L260F) was produced in the clostridial expression system. Comparative analyses of the
in vitro
, cellular, and
in vivo
activities of rBoNT/A4 and rBoNT/A4-L260F I264R showed 1,000-fold-lower activity than BoNT/A1 in both the mutated and nonmutated BoNT/A4. This indicates that these mutations do not alter the activity of BoNT/A4 holotoxin. In summary, a recombinant BoNT from a dual-toxin-producing strain was expressed and purified in an endogenous clostridial expression system, allowing analysis of this toxin.
Routes of Acquisition of the Gut Microbiota of the Honey Bee Apis mellifera
Authors: Powell J. Elijah , Martinson Vincent G. , Urban-Mead Katherine , Moran Nancy A. 
Published: September 2014

ABSTRACT

Studies of newly emerged
Apis mellifera
worker bees have demonstrated that their guts are colonized by a consistent core microbiota within several days of eclosure. We conducted experiments aimed at illuminating the transmission routes and spatiotemporal colonization dynamics of this microbiota. Experimental groups of newly emerged workers were maintained in cup cages and exposed to different potential transmission sources. Colonization patterns were evaluated using quantitative real-time PCR (qPCR) to assess community sizes and using deep sequencing of 16S rRNA gene amplicons to assess community composition. In addition, we monitored the establishment of the ileum and rectum communities within workers sampled over time from natural hive conditions. The study verified that workers initially lack gut bacteria and gain large characteristic communities in the ileum and rectum within 4 to 6 days within hives. Typical communities, resembling those of workers within hives, were established in the presence of nurse workers or nurse worker fecal material, and atypical communities of noncore or highly skewed compositions were established when workers were exposed only to oral trophallaxis or hive components (comb, honey, bee bread). The core species of Gram-negative bacteria,
Snodgrassella alvi
,
Gilliamella apicola
, and
Frischella perrara
, were dependent on the presence of nurses or hindgut material, whereas some Gram-positive species were more often transferred through exposure to hive components. These results indicate aspects of the colony life cycle and behavior that are key to the propagation of the characteristic honey bee gut microbiota.
Expanding the Halohydrin Dehalogenase Enzyme Family: Identification of Novel Enzymes by Database Mining
Authors: Schallmey Marcus , Koopmeiners Julia , Wells Elizabeth , Wardenga Rainer , Schallmey Anett 
Published: September 2014

ABSTRACT

Halohydrin dehalogenases are very rare enzymes that are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described that have led to several interesting and industrially important applications. Nevertheless, only a few of these enzymes have been made available through recombinant techniques; hence, it is of general interest to expand the repertoire of these enzymes so as to enable novel biocatalytic applications. After the identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes that could be heterologously expressed also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short-chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different from previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined, greatly expanding the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for
in silico
identification of very rare but biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever-growing sequence databases and, as a consequence, make even more members of this interesting enzyme family available to the scientific and industrial community.
Prebiotic Oligosaccharides: Comparative Evaluation Using
In Vitro
Cultures of Infants' Fecal Microbiomes
Authors: Stiverson J. , Williams T. , Chen J. , Adams S. , Hustead D. , Price P. , Guerrieri J. , Deacon J. , Yu Z. 
Published: September 2014

ABSTRACT

The objective of this study was to systematically assess the bifidogenic effect of three commonly used prebiotic products using
in vitro
cultures of infant fecal samples. Fresh stool samples collected from six term infants, each exclusively fed human milk (
n
= 3) or infant formula (
n
= 3), at 28 days of age were used as inocula. The following prebiotic products were added at concentrations applicable to infant formula: Vivinal GOS 15 (containing 28.5% galacto-oligosaccharide [GOS]) at 7.2 g/liter, Beneo HP (99.5% long-chain inulin [IN]) at 0.8 g/liter, Beneo Synergy 1 (enriched oligofructose and inulin [OF-IN]) at 4 g/liter, and a combination of Vivinal GOS 15 (7.2 g/liter) and Beneo HP (0.8 g/liter) (GOS-IN). The growth of total bacteria,
Bifidobacterium
,
Bacteroides
,
Bifidobacterium longum
, and
Escherichia coli
was quantified using specific quantitative PCR (qPCR).
Bifidobacterium
was also enumerated on selective Beerens agar plates, with representative colonies identified by sequencing of their 16S rRNA genes. Volatile fatty acids (VFA) and pH in the cultures were also determined. Irrespective of the feeding methods, the GOS product, either alone or in combination with Beneo HP, resulted in substantially higher growth of total bifidobacteria, and much of this growth was attributed to growth of
B. longum
. Beneo Synergy 1 also increased the abundance of total bifidobacteria and
B. longum
. Corresponding to the increases in these two bacterial groups, acetic acid concentrations were higher, while there was a trend of lower
E. coli
levels and pH. The lower pH and higher acetic acid concentration might be directly responsible for the lower
E. coli
population. At the concentrations studied, the GOS product was more bifidogenic and potent in inhibiting
E. coli
than the other products tested. These results suggest that supplementation of infant formula with GOS may increase intestinal bifidobacteria and benefit infant health.
Microbial Synthesis of the Forskolin Precursor Manoyl Oxide in an Enantiomerically Pure Form
Authors: Nielsen Morten T. , Ranberg Johan Andersen , Christensen Ulla , Christensen Hanne Bjerre , Harrison Scott J. , Olsen Carl Erik , Hamberger Björn , Møller Birger Lindberg , Nørholm Morten H. H. 
Published: September 2014

ABSTRACT

Forskolin is a promising medicinal compound belonging to a plethora of specialized plant metabolites that constitute a rich source of bioactive high-value compounds. A major obstacle for exploitation of plant metabolites is that they often are produced in small amounts and in plants difficult to cultivate. This may result in insufficient and unreliable supply leading to fluctuating and high sales prices. Hence, substantial efforts and resources have been invested in developing sustainable and reliable supply routes based on microbial cell factories. Here, we report microbial synthesis of (13
R
)-manoyl oxide, a proposed intermediate in the biosynthesis of forskolin and other medically important labdane-type terpenoids. Process optimization enabled synthesis of enantiomerically pure (13
R
)-manoyl oxide as the sole metabolite, providing a pure compound in just two steps with a yield of 10 mg/liter. The work presented here demonstrates the value of a standardized bioengineering pipeline and the large potential of microbial cell factories as sources for sustainable synthesis of complex biochemicals.
Microbiota in the Throat and Risk Factors for Laryngeal Carcinoma
Authors: Gong Hongli , Shi Yi , Zhou Xia , Wu Chunping , Cao Pengyu , Xu Chen , Hou Dongsheng , Wang Yuezhu , Zhou Liang 
Published: September 2014

ABSTRACT

The compositions and abundances of the microbiota in the ecological niche of the human throat and the possible relationship between the microbiota and laryngeal cancer are poorly understood. To obtain insight into this, we enrolled 27 laryngeal carcinoma patients and 28 subjects with vocal cord polyps as controls. For each subject, we simultaneously collected swab samples from the upper throat near the epiglottis (site I) and tissue samples from the vestibulum laryngis to the subglottic region (site II). The microbiota of the throat were fully characterized by pyrosequencing of barcoded 16S rRNA genes. We found 14 phyla, 20 classes, 38 orders, 85 families, and 218 genera in the throats of enrolled subjects. The main phyla were
Firmicutes
(54.7%),
Fusobacteria
(14.8%),
Bacteroidetes
(12.7%), and
Proteobacteria
(10.6%).
Streptococcus
(37.3%),
Fusobacterium
(11.3%), and
Prevotella
(10.6%) were identified as the three most predominant genera in the throat. The relative abundances of 23 bacterial genera in site I were significantly different from those in site II (
P
< 0.05). The relative proportions of 12 genera largely varied between laryngeal cancer patients and control subjects (
P
< 0.05). Collectively, this study outlined the spatial structure of microbial communities in the human throat. The spatial structure of bacterial communities significantly varied in two anatomical sites of the throat. The bacterial profiles of the throat of laryngeal cancer patients were strongly different from those of control subjects, and several of these microorganisms may be related to laryngeal carcinoma.
Conversion of the Pseudomonas aeruginosa Quinolone Signal and Related Alkylhydroxyquinolines by Rhodococcus sp. Strain BG43
Authors: Müller Christine , Birmes Franziska S. , Niewerth Heiko , Fetzner Susanne 
Published: September 2014

ABSTRACT

A bacterial strain, which based on the sequences of its 16S rRNA,
gyrB
,
catA
, and
qsdA
genes, was identified as a
Rhodococcus
sp. closely related to
Rhodococcus erythropolis
, was isolated from soil by enrichment on the
Pseudomonas
quinolone signal [PQS; 2-heptyl-3-hydroxy-4(1
H
)-quinolone], a quorum sensing signal employed by the opportunistic pathogen
Pseudomonas aeruginosa
. The isolate, termed
Rhodococcus
sp. strain BG43, cometabolically degraded PQS and its biosynthetic precursor 2-heptyl-4(1
H
)-quinolone (HHQ) to anthranilic acid. HHQ degradation was accompanied by transient formation of PQS, and HHQ hydroxylation by cell extracts required NADH, indicating that strain BG43 has a HHQ monooxygenase isofunctional to the biosynthetic enzyme PqsH of
P. aeruginosa
. The enzymes catalyzing HHQ hydroxylation and PQS degradation were inducible by PQS, suggesting a specific pathway. Remarkably,
Rhodococcus
sp. BG43 is also capable of transforming 2-heptyl-4-hydroxyquinoline-
N
-oxide to PQS. It thus converts an antibacterial secondary metabolite of
P. aeruginosa
to a quorum sensing signal molecule.
Fine-Tuning of the Fatty Acid Pathway by Synthetic Antisense RNA for Enhanced (2
S
)-Naringenin Production from
l
-Tyrosine in Escherichia coli
Authors: Wu Junjun , Yu Oliver , Du Guocheng , Zhou Jingwen , Chen Jian 
Published: September 2014

ABSTRACT

Malonyl coenzyme A (malonyl-CoA) is an important precursor for the synthesis of natural products, such as polyketides and flavonoids. The majority of this cofactor often is consumed for producing fatty acids and phospholipids, leaving only a small amount of cellular malonyl-CoA available for producing the target compound. The tuning of malonyl-CoA into heterologous pathways yields significant phenotypic effects, such as growth retardation and even cell death. In this study, fine-tuning of the fatty acid pathway in
Escherichia coli
with antisense RNA (asRNA) to balance the demands on malonyl-CoA for target-product synthesis and cell health was proposed. To establish an efficient asRNA system, the relationship between sequence and function for asRNA was explored. It was demonstrated that the gene-silencing effect of asRNA could be tuned by directing asRNA to different positions in the 5′-UTR (untranslated region) of the target gene. Based on this principle, the activity of asRNA was quantitatively tailored to balance the need for malonyl-CoA in cell growth and the production of the main flavonoid precursor, (2
S
)-naringenin. Appropriate inhibitory efficiency of the anti-
fabB
/
fabF
asRNA improved the production titer by 431% (391 mg/liter). Therefore, the strategy presented in this study provided a useful tool for the fine-tuning of endogenous gene expression in bacteria.
Novel Broad-Spectrum Antimicrobial Photoinactivation of
In Situ
Oral Biofilms by Visible Light plus Water-Filtered Infrared A
Authors: Karygianni L. , Ruf S. , Follo M. , Hellwig E. , Bucher M. , Anderson A. C. , Vach K. , Al-Ahmad A. 
Published: September 2014

ABSTRACT

Antimicrobial photodynamic therapy (APDT) has gained increased attention as an alternative treatment approach in various medical fields. However, the effect of APDT using visible light plus water-filtered infrared A (VIS + wIRA) on oral biofilms remains unexplored. For this purpose, initial and mature oral biofilms were obtained
in situ
; six healthy subjects wore individual upper jaw acrylic devices with bovine enamel slabs attached to their proximal sites for 2 h or 3 days. The biofilms were incubated with 100 μg ml
−1
toluidine blue O (TB) or chlorin e6 (Ce6) and irradiated with VIS + wIRA with an energy density of 200 mW cm
−2
for 5 min. After cultivation, the CFU of half of the treated biofilm samples were quantified, whereas following live/dead staining, the other half of the samples were monitored by confocal laser scanning microscopy (CLSM). TB- and Ce6-mediated APDT yielded a significant decrease of up to 3.8 and 5.7 log
10
CFU for initial and mature oral biofilms, respectively. Quantification of the stained photoinactivated microorganisms confirmed these results. Overall, CLSM revealed the diffusion of the tested photosensitizers into the deepest biofilm layers after exposure to APDT. In particular, Ce6-aided APDT presented elevated permeability and higher effectiveness in eradicating 89.62% of biofilm bacteria compared to TB-aided APDT (82.25%) after 3 days. In conclusion, antimicrobial photoinactivation using VIS + wIRA proved highly potent in eradicating oral biofilms. Since APDT excludes the development of microbial resistance, it could supplement the pharmaceutical treatment of periodontitis or peri-implantitis.
Trimethylamine and Organic Matter Additions Reverse Substrate Limitation Effects on the δ
13
C Values of Methane Produced in Hypersaline Microbial Mats
Authors: Kelley Cheryl A. , Nicholson Brooke E. , Beaudoin Claire S. , Detweiler Angela M. , Bebout Brad M. 
Published: September 2014

ABSTRACT

Methane production has been observed in a number of hypersaline environments, and it is generally thought that this methane is produced through the use of noncompetitive substrates, such as the methylamines, dimethylsulfide and methanol. Stable isotope measurements of the produced methane have also suggested that the methanogens are operating under conditions of substrate limitation. Here, substrate limitation in gypsum-hosted endoevaporite and soft-mat hypersaline environments was investigated by the addition of trimethylamine, a noncompetitive substrate for methanogenesis, and dried microbial mat, a source of natural organic matter. The δ
13
C values of the methane produced after amendments were compared to those in unamended control vials. At all hypersaline sites investigated, the δ
13
C values of the methane produced in the amended vials were statistically lower (by 10 to 71‰) than the unamended controls, supporting the hypothesis of substrate limitation at these sites. When substrates were added to the incubation vials, the methanogens within the vials fractionated carbon isotopes to a greater degree, resulting in the production of more
13
C-depleted methane. Trimethylamine-amended samples produced lower methane δ
13
C values than the mat-amended samples. This difference in the δ
13
C values between the two types of amendments could be due to differences in isotope fractionation associated with the dominant methane production pathway (or substrate used) within the vials, with trimethylamine being the main substrate used in the trimethylamine-amended vials. It is hypothesized that increased natural organic matter in the mat-amended vials would increase fermentation rates, leading to higher H
2
concentrations and increased CO
2
/H
2
methanogenesis.
Activities of Alkyl Hydroperoxide Reductase Subunits C1 and C2 of Vibrio parahaemolyticus against Different Peroxides
Authors: Chung Chun-Hui , Ma Tsung-yong , Fen Shin-yuan , Wong Hin-chung 
Published: September 2014

ABSTRACT

Alkyl hydroperoxide reductase subunit C gene (
ahpC
) functions were characterized in
Vibrio parahaemolyticus
, a commonly occurring marine food-borne enteropathogenic bacterium. Two
ahpC
genes,
ahpC1
(VPA1683) and
ahpC2
(VP0580), encoded putative two-cysteine peroxiredoxins, which are highly similar to the homologous proteins of
Vibrio vulnificus
. The responses of deletion mutants of
ahpC
genes to various peroxides were compared with and without gene complementation and at different incubation temperatures. The growth of the
ahpC1
mutant and
ahpC1 ahpC2
double mutant in liquid medium was significantly inhibited by organic peroxides, cumene hydroperoxide and
tert
-butyl hydroperoxide. However, inhibition was higher at 12°C and 22°C than at 37°C. Inhibiting effects were prevented by the complementary
ahpC1
gene. Inconsistent detoxification of H
2
O
2
by
ahpC
genes was demonstrated in an agar medium but not in a liquid medium. Complementation with an
ahpC2
gene partially restored the peroxidase effect in the double
ahpC1 ahpC2
mutant at 22°C. This investigation reveals that
ahpC1
is the chief peroxidase gene that acts against organic peroxides in
V. parahaemolyticus
and that the function of the
ahpC
genes is influenced by incubation temperature.