bluuebunny/crossref_metadata_2025_split huggingface dataset:
Metabolism of 2-Methylpropene (Isobutylene) by the Aerobic Bacterium Mycobacterium sp. Strain ELW1
Authors: Kottegoda Samanthi , Waligora Elizabeth , Hyman Michael 
Published: January 2015

ABSTRACT

An aerobic bacterium (
Mycobacterium
sp. strain ELW1) that utilizes 2-methylpropene (isobutylene) as a sole source of carbon and energy was isolated and characterized. Strain ELW1 grew on 2-methylpropene (growth rate = 0.05 h
−1
) with a yield of 0.38 mg (dry weight) mg 2-methylpropene
−1
. Strain ELW1 also grew more slowly on both
cis
- and
trans
-2-butene but did not grow on any other C
2
to C
5
straight-chain, branched, or chlorinated alkenes tested. Resting 2-methylpropene-grown cells consumed ethene, propene, and 1-butene without a lag phase. Epoxyethane accumulated as the only detected product of ethene oxidation. Both alkene consumption and epoxyethane production were fully inhibited in cells exposed to 1-octyne, suggesting that alkene oxidation is initiated by an alkyne-sensitive, epoxide-generating monooxygenase. Kinetic analyses indicated that 1,2-epoxy-2-methylpropane is rapidly consumed during 2-methylpropene degradation, while 2-methyl-2-propen-1-ol is not a significant metabolite of 2-methylpropene catabolism. Degradation of 1,2-epoxy-2-methylpropane by 2-methylpropene-grown cells led to the accumulation and further degradation of 2-methyl-1,2-propanediol and 2-hydroxyisobutyrate, two sequential metabolites previously identified in the aerobic microbial metabolism of methyl
tert
-butyl ether (MTBE) and
tert
-butyl alcohol (TBA). Growth of strain ELW1 on 2-methylpropene, 1,2-epoxy-2-methylpropane, 2-methyl-1,2-propanediol, and 2-hydroxyisobutyrate was fully inhibited when cobalt ions were omitted from the growth medium, while growth on 3-hydroxybutyrate and other substrates was unaffected by the absence of added cobalt ions. Our results suggest that, like aerobic MTBE- and TBA-metabolizing bacteria, strain ELW1 utilizes a cobalt/cobalamin-dependent mutase to transform 2-hydroxyisobutyrate. Our results have been interpreted in terms of their impact on our understanding of the microbial metabolism of alkenes and ether oxygenates.
Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase
Authors: Komati Reddy Gajendar , Lindner Steffen N. , Wendisch Volker F. 
Published: January 2015

ABSTRACT

Corynebacterium glutamicum
uses the Embden-Meyerhof-Parnas pathway of glycolysis and gains 2 mol of ATP per mol of glucose by substrate-level phosphorylation (SLP). To engineer glycolysis without net ATP formation by SLP, endogenous phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was replaced by nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) from
Clostridium acetobutylicum
, which irreversibly converts glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) without generating ATP. As shown recently (S. Takeno, R. Murata, R. Kobayashi, S. Mitsuhashi, and M. Ikeda, Appl Environ Microbiol 76:7154–7160, 2010,
http://dx.doi.org/10.1128/AEM.01464-10
), this ATP-neutral, NADPH-generating glycolytic pathway did not allow for the growth of
Corynebacterium glutamicum
with glucose as the sole carbon source unless hitherto unknown suppressor mutations occurred; however, these mutations were not disclosed. In the present study, a suppressor mutation was identified, and it was shown that heterologous expression of
udhA
encoding soluble transhydrogenase from
Escherichia coli
partly restored growth, suggesting that growth was inhibited by NADPH accumulation. Moreover, genome sequence analysis of second-site suppressor mutants that were able to grow faster with glucose revealed a single point mutation in the gene of non-proton-pumping NADH:ubiquinone oxidoreductase (NDH-II) leading to the amino acid change D213G, which was shared by these suppressor mutants. Since related NDH-II enzymes accepting NADPH as the substrate possess asparagine or glutamine residues at this position, D213G, D213N, and D213Q variants of
C. glutamicum
NDH-II were constructed and were shown to oxidize NADPH in addition to NADH. Taking these findings together, ATP-neutral glycolysis by the replacement of endogenous NAD-dependent GAPDH with NADP-dependent GapN became possible via oxidation of NADPH formed in this pathway by mutant NADPH-accepting NDH-II
D213G
and thus by coupling to electron transport phosphorylation (ETP).
Oligopolyphenylenevinylene-Conjugated Oligoelectrolyte Membrane Insertion Molecules Selectively Disrupt Cell Envelopes of Gram-Positive Bacteria
Authors: Hinks Jamie , Poh Wee Han , Chu Justin Jang Hann , Loo Joachim Say Chye , Bazan Guillermo C. , Hancock Lynn E. , Wuertz Stefan 
Published: January 2015

ABSTRACT

The modification of microbial membranes to achieve biotechnological strain improvement with exogenous small molecules, such as oligopolyphenylenevinylene-conjugated oligoelectrolyte (OPV-COE) membrane insertion molecules (MIMs), is an emerging biotechnological field. Little is known about the interactions of OPV-COEs with their target, the bacterial envelope. We studied the toxicity of three previously reported OPV-COEs with a selection of Gram-negative and Gram-positive organisms and demonstrated that Gram-positive bacteria are more sensitive to OPV-COEs than Gram-negative bacteria. Transmission electron microscopy demonstrated that these MIMs disrupt microbial membranes and that this occurred to a much greater degree in Gram-positive organisms. We used a number of mutants to probe the nature of MIM interactions with the microbial envelope but were unable to align the membrane perturbation effects of these compounds to previously reported membrane disruption mechanisms of, for example, cationic antimicrobial peptides. Instead, the data support the notion that OPV-COEs disrupt microbial membranes through a suspected interaction with diphosphatidylglycerol (DPG), a major component of Gram-positive membranes. The integrity of model membranes containing elevated amounts of DPG was disrupted to a greater extent by MIMs than those prepared from
Escherichia coli
total lipid extracts alone.
Distinct Roles for Carbohydrate-Binding Modules of Glycoside Hydrolase 10 (GH10) and GH11 Xylanases from Caldicellulosiruptor sp. Strain F32 in Thermostability and Catalytic Efficiency
Authors: Meng Dong-Dong , Ying Yu , Chen Xiao-Hua , Lu Ming , Ning Kang , Wang Lu-Shan , Li Fu-Li 
Published: January 2015

ABSTRACT

Xylanases are crucial for lignocellulosic biomass deconstruction and generally contain noncatalytic carbohydrate-binding modules (CBMs) accessing recalcitrant polymers. Understanding how multimodular enzymes assemble can benefit protein engineering by aiming at accommodating various environmental conditions. Two multimodular xylanases, XynA and XynB, which belong to glycoside hydrolase families 11 (GH11) and GH10, respectively, have been identified from
Caldicellulosiruptor
sp. strain F32. In this study, both xylanases and their truncated mutants were overexpressed in
Escherichia coli
, purified, and characterized. GH11 XynATM1 lacking CBM exhibited a considerable improvement in specific activity (215.8 U nmol
−1
versus 94.7 U nmol
−1
) and thermal stability (half-life of 48 h versus 5.5 h at 75°C) compared with those of XynA. However, GH10 XynB showed higher enzyme activity and thermostability than its truncated mutant without CBM. Site-directed mutagenesis of N-terminal amino acids resulted in a mutant, XynATM1-M, with 50% residual activity improvement at 75°C for 48 h, revealing that the disordered region influenced protein thermostability negatively. The thermal stability of both xylanases and their truncated mutants were consistent with their melting temperature (

T
m

), which was determined by using differential scanning calorimetry. Through homology modeling and cross-linking analysis, we demonstrated that for XynB, the resistance against thermoinactivation generally was enhanced through improving both domain properties and interdomain interactions, whereas for XynA, no interdomain interactions were observed. Optimized intramolecular interactions can accelerate thermostability, which provided microbes a powerful evolutionary strategy to assemble catalysts that are adapted to various ecological conditions.
Inhibitory Effects of C
2
to C
10
1-Alkynes on Ammonia Oxidation in Two Nitrososphaera Species
Authors: Taylor A. E. , Taylor K. , Tennigkeit B. , Palatinszky M. , Stieglmeier M. , Myrold D. D. , Schleper C. , Wagner M. , Bottomley P. J. 
Published: January 2015

ABSTRACT

A previous study showed that ammonia oxidation by the
Thaumarchaeota
Nitrosopumilus maritimus
(group 1.1a) was resistant to concentrations of the C
8
1-alkyne, octyne, which completely inhibits activity by ammonia-oxidizing bacteria. In this study, the inhibitory effects of octyne and other C
2
to C
10
1-alkynes were evaluated on the nitrite production activity of two pure culture isolates from
Thaumarchaeota
group 1.1b,
Nitrososphaera viennensis
strain EN76 and
Nitrososphaera gargensis
. Both
N. viennensis
and
N. gargensis
were insensitive to concentrations of octyne that cause complete and irreversible inactivation of nitrite production by ammonia-oxidizing bacteria. However, octyne concentrations (≥20 μM) that did not inhibit
N. maritimus
partially inhibited nitrite production in
N. viennensis
and
N. gargensis
in a manner that did not show the characteristics of irreversible inactivation. In contrast to previous studies with an ammonia-oxidizing bacterium,
Nitrosomonas europaea
, octyne inhibition of
N. viennensis
was: (i) fully and immediately reversible, (ii) not competitive with NH
4
+
, and (iii) without effect on the competitive interaction between NH
4
+
and acetylene. Both
N. viennensis
and
N. gargensis
demonstrated the same overall trend in regard to 1-alkyne inhibition as previously observed for
N. maritimus
, being highly sensitive to ≤C
5
alkynes and more resistant to longer-chain length alkynes. Reproducible differences were observed among
N. maritimus
,
N. viennensis
, and
N. gargensis
in regard to the extent of their resistance/sensitivity to C
6
and C
7
1-alkynes, which may indicate differences in the ammonia monooxygenase binding and catalytic site(s) among the
Thaumarchaeota
.
Genetic Determinants of Reutericyclin Biosynthesis in Lactobacillus reuteri
Authors: Lin Xiaoxi B. , Lohans Christopher T. , Duar Rebbeca , Zheng Jinshui , Vederas John C. , Walter Jens , Gänzle Michael 
Published: January 2015

ABSTRACT

Reutericyclin is a unique antimicrobial tetramic acid produced by some strains of
Lactobacillus reuteri
. This study aimed to identify the genetic determinants of reutericyclin biosynthesis. Comparisons of the genomes of reutericyclin-producing
L. reuteri
strains with those of non-reutericyclin-producing strains identified a genomic island of 14 open reading frames (ORFs) including genes coding for a nonribosomal peptide synthetase (NRPS), a polyketide synthase (PKS), homologues of PhlA, PhlB, and PhlC, and putative transport and regulatory proteins. The protein encoded by
rtcN
is composed of a condensation domain, an adenylation domain likely specific for
d
-leucine, and a thiolation domain.
rtcK
codes for a PKS that is composed of a ketosynthase domain, an acyl-carrier protein domain, and a thioesterase domain. The products of
rtcA
,
rtcB
, and
rtcC
are homologous to the diacetylphloroglucinol-biosynthetic proteins PhlABC and may acetylate the tetramic acid moiety produced by RtcN and RtcK, forming reutericyclin. Deletion of
rtcN
or
rtcABC
in
L. reuteri
TMW1.656 abrogated reutericyclin production but did not affect resistance to reutericyclin. Genes coding for transport and regulatory proteins could be deleted only in the reutericyclin-negative
L. reuteri
strain TMW1.656Δ
rtcN
, and these deletions eliminated reutericyclin resistance. The genomic analyses suggest that the reutericyclin genomic island was horizontally acquired from an unknown source during a unique event. The combination of PhlABC homologues with both an NRPS and a PKS has also been identified in the lactic acid bacteria
Streptococcus mutans
and
Lactobacillus plantarum
, suggesting that the genes in these organisms and those in
L. reuteri
share an evolutionary origin.
AmyM, a Novel Maltohexaose-Forming α-Amylase from Corallococcus sp. Strain EGB
Authors: Li Zhoukun , Wu Jiale , Zhang Biying , Wang Fei , Ye Xianfeng , Huang Yan , Huang Qiang , Cui Zhongli 
Published: January 2015

ABSTRACT

A novel α-amylase, AmyM, was purified from the culture supernatant of
Corallococcus
sp. strain EGB. AmyM is a maltohexaose-forming exoamylase with an apparent molecular mass of 43 kDa. Based on the results of matrix-assisted laser desorption ionization–time of flight mass spectrometry and peptide mass fingerprinting of AmyM and by comparison to the genome sequence of
Corallococcus
coralloides
DSM 2259, the AmyM gene was identified and cloned into
Escherichia coli
.
amyM
encodes a secretory amylase with a predicted signal peptide of 23 amino acid residues, which showed no significant identity with known and functionally verified amylases.
amyM
was expressed in
E. coli
BL21(DE3) cells with a hexahistidine tag. The signal peptide efficiently induced the secretion of mature AmyM in
E. coli
. Recombinant AmyM (rAmyM) was purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography, with a specific activity of up to 14,000 U/mg. rAmyM was optimally active at 50°C in Tris-HCl buffer (50 mM; pH 7.0) and stable at temperatures of <50°C. rAmyM was stable over a wide range of pH values (from pH 5.0 to 10.0) and highly tolerant to high concentrations of salts, detergents, and various organic solvents. Its activity toward starch was independent of calcium ions. The

K
m

and
V
max
of recombinant AmyM for soluble starch were 6.61 mg ml
−1
and 44,301.5 μmol min
−1
mg
−1
, respectively. End product analysis showed that maltohexaose accounted for 59.4% of the maltooligosaccharides produced. These characteristics indicate that AmyM has great potential in industrial applications.
DNA Double-Strand Breaks and Telomeres Play Important Roles in Trypanosoma brucei Antigenic Variation
Authors: Li Bibo 
Published: January 2015

ABSTRACT

Human-infecting microbial pathogens all face a serious problem of elimination by the host immune response. Antigenic variation is an effective immune evasion mechanism where the pathogen regularly switches its major surface antigen. In many cases, the major surface antigen is encoded by genes from the same gene family, and its expression is strictly monoallelic. Among pathogens that undergo antigenic variation,
Trypanosoma brucei
(a kinetoplastid), which causes human African trypanosomiasis,
Plasmodium falciparum
(an apicomplexan), which causes malaria,
Pneumocystis jirovecii
(a fungus), which causes pneumonia, and
Borrelia burgdorferi
(a bacterium), which causes Lyme disease, also express their major surface antigens from loci next to the telomere. Except for
Plasmodium
, DNA recombination-mediated gene conversion is a major pathway for surface antigen switching in these pathogens. In the last decade, more sophisticated molecular and genetic tools have been developed in
T. brucei
, and our knowledge of functions of DNA recombination in antigenic variation has been greatly advanced. VSG is the major surface antigen in
T. brucei
. In subtelomeric VSG expression sites (ESs),
VSG
genes invariably are flanked by a long stretch of upstream 70-bp repeats. Recent studies have shown that DNA double-strand breaks (DSBs), particularly those in 70-bp repeats in the active ES, are a natural potent trigger for antigenic variation in
T. brucei
. In addition, telomere proteins can influence VSG switching by reducing the DSB amount at subtelomeric regions. These findings will be summarized and their implications will be discussed in this review.
Pilot test of the Healthy Food Environment Policy Index (Food-EPI) to increase government actions for creating healthy food environments
Authors: Vandevijvere Stefanie , Swinburn Boyd 
Published: January 2015

ObjectivesEffective government policies are essential to increase the healthiness of food environments. The International Network for Food and Obesity/non-communicable diseases (NCDs) Research, Monitoring and Action Support (INFORMAS) has developed a monitoring tool (the Healthy Food Environment Policy Index (Food-EPI)) and process to rate government policies to create healthy food environments against international best practice. The aims of this study were to pilot test the Food-EPI, and revise the tool and process for international implementation.SettingNew Zealand.ParticipantsThirty-nine informed, independent public health experts and non-governmental organisation (NGO) representatives.Primary and secondary outcome measuresEvidence on the extent of government implementation of different policies on food environments and infrastructure support was collected in New Zealand and validated with government officials. Two whole-day workshops were convened of public health experts and NGO representatives who rated performance of their government for seven policy and seven infrastructure support domains against international best practice. In addition, the raters evaluated the level of difficulty of rating, and appropriateness and completeness of the evidence presented for each indicator.ResultsInter-rater reliability was 0.85 (95% CI 0.81 to 0.88; Gwet’s AC2) using quadratic weights, and increased to 0.89 (95% CI 0.85 to 0.92) after deletion of the problematic indicators. Based on raters’ assessments and comments, major changes to the Food-EPI tool include strengthening the leadership domain, removing the workforce development domain, a stronger focus on equity, and adding community-based programmes and government funding for research on obesity and diet-related NCD prevention, as good practice indicators.ConclusionsThe resulting tool and process will be promoted and offered to countries of varying size and income globally. International benchmarking of the extent of government policy implementation on food environments has the potential to catalyse greater government action to reduce obesity and NCDs, and increase civil society's capacity to advocate for healthy food environments.
Protocol for the surgical and large bore procedures in malignant pleural mesothelioma and radiotherapy trial (SMART Trial): an RCT evaluating whether prophylactic radiotherapy reduces the incidence of procedure tract metastases
Authors: Clive Amelia O , Wilson Paula , Taylor Hazel , Morley Anna J , de Winton Emma , Panakis Niki , Rahman Najib , Pepperell Justin , Howell Timothy , Batchelor Timothy J P , Jordan Nikki , Lee Y C Gary , Dobson Lee , Maskell Nick A 
Published: January 2015

IntroductionPatients with malignant pleural mesothelioma (MPM) may develop painful ‘procedure tract metastasis’ (PTM) at the site of previous pleural interventions. Prophylactic radiotherapy has been used to minimise this complication; however, three small randomised trials have shown conflicting results regarding its effectiveness. The surgical and large bore procedures in malignant pleural mesothelioma and radiotherapy trial (SMART Trial) is a suitably powered, multicentre, randomised controlled trial, designed to evaluate the efficacy of prophylactic radiotherapy within 42 days of pleural instrumentation in preventing the development of PTM in MPM.Methods and analysis203 patients with a histocytologically proven diagnosis of MPM, who have undergone a large bore pleural intervention (thoracic surgery, large bore chest drain, indwelling pleural catheter or local anaesthetic thoracoscopy) in the previous 35 days, will be recruited from UK hospitals. Patients will be randomised (1:1) to receive immediate radiotherapy (21 Gy in 3 fractions over 3 working days within 42 days of the pleural intervention) or deferred radiotherapy (21 Gy in 3 fractions over 3 working days given if a PTM develops). Patients will be followed up for 12 months. The primary outcome measure is the rate of PTM until death or 12 months (whichever is sooner), as defined by the presence of a clinically palpable nodule of at least 1 cm diameter felt within 7 cm of the margins of the procedure site as confirmed by two assessors. Secondary outcome measures include chest pain, quality of life, analgaesic requirements, healthcare utilisation and safety (including radiotherapy toxicity).Ethics and disseminationThe trial has received ethical approval from the Southampton B Research Ethics Committee (11/SC/0408). There is a Trial Steering Committee, including independent members and a patient and public representative. The trial results will be published in a peer-reviewed journal and presented at international conferences.Trial registration numberISRCTN72767336.
A short-term risk–benefit analysis of occasional and regular use of low-dose aspirin in primary prevention of vascular diseases: a nationwide population-based study
Authors: Wu I-Chen , Hsieh Hui-Min , Wu Ming-Tsang 
Published: January 2015

ObjectiveTo calculate the short-term risk–benefit effect of occasional and regular use of low-dose aspirin (≤100 mg/day) in primary prevention.Study designTwo retrospective cohort studies.SettingTaiwan.Participants63 788 and 24 910 patients of two nationwide population-based studies were examined.MethodsTwo databases of 1 000 000 patients were randomly sampled from data of Taiwan's National Health Insurance (NHI) for years 1997–2000 (NHI 2000) and 2005 (NHI 2005). In NHI 2000, 63 788 patients 30–95 years of age were found not to have previously been prescribed aspirin before 1 January 2000, but to have first been prescribed low-dose aspirin after that date. They were also found to be at risk of first hospitalisation for any major vascular diseases including haemorrhage (major gastrointestinal haemorrhage or cerebral haemorrhage) and ischaemia (acute myocardial infarction or ischaemic stroke) after their first prescription. We also applied it to NHI 2005, and the number of eligible patients was 24 910. Patients prescribed low-dose aspirin for <20% of the days of a 60-day follow-up period were considered to be occasional users, and those prescribed low-dose aspirin for ≥80% of the days were considered to be regular users. Differences in rate of haemorrhage and ischaemia between these users were used to calculate their net clinical risk.Primary outcomeVascular diseases.ResultsIn NHI 2000, the overall unadjusted rates of haemorrhage and ischaemia were 0.09% and 0.21%, respectively, for occasional users and 0.32% and 2.30%, respectively, for regular users. Adjusted net clinical risk of low-dose aspirin use between the two groups was 2.24% (95% CI 2.03% to 2.48%; p<0.001). Similar results were also found in NHI 2005.ConclusionsShort-term regular use of low-dose aspirin might not be better than occasional use for preventing major vascular diseases in primary prevention. Prescribing regular low-dose aspirin for primary prevention should be done with caution. Future studies should explore the risk–benefit effect of long-term low-dose aspirin use in primary prevention.