bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intracellular and Extracellular Expression of Bacillus thuringiensis Crystal Protein Cry5B in Lactococcus lactis for Use as an Anthelminthic
Authors: Durmaz Evelyn , Hu Yan , Aroian Raffi V. , Klaenhammer Todd R. 
Published: December 2015

ABSTRACT

The
Bacillus thuringiensis
crystal (Cry) protein Cry5B (140 kDa) and a truncated version of the protein, tCry5B (79 kDa), are lethal to nematodes. Genes encoding the two proteins were separately cloned into a high-copy-number vector with a strong constitutive promoter (pTRK593) in
Lactococcus lactis
for potential oral delivery against parasitic nematode infections. Western blots using a Cry5B-specific antibody revealed that constitutively expressed Cry5B and tCry5B were present in both cells and supernatants. To increase production,
cry5B
was cloned into the high-copy-number plasmid pMSP3535H3, carrying a nisin-inducible promoter. Immunoblotting revealed that 3 h after nisin induction, intracellular Cry5B was strongly induced at 200 ng/ml nisin, without adversely affecting cell viability or cell membrane integrity. Both Cry5B genes were also cloned into plasmid pTRK1061, carrying a promoter and encoding a transcriptional activator that invoke low-level expression of prophage holin and lysin genes in
Lactococcus
lysogens, resulting in a leaky phenotype. Cry5B and tCry5B were actively expressed in the lysogenic strain
L. lactis
KP1 and released into cell supernatants without affecting culture growth. Lactate dehydrogenase (LDH) assays indicated that Cry5B, but not LDH, leaked from the bacteria. Lastly, using intracellular lysates from
L. lactis
cultures expressing both Cry5B and tCry5B,
in vivo
challenges of
Caenorhabditis elegans
worms demonstrated that the Cry proteins were biologically active. Taken together, these results indicate that active Cry5B proteins can be expressed intracellularly in and released extracellularly from
L. lactis
, showing potential for future use as an anthelminthic that could be delivered orally in a food-grade microbe.
Comparison of Four Comamonas Catabolic Plasmids Reveals the Evolution of pBHB To Catabolize Haloaromatics
Authors: Chen Kai , Xu Xihui , Zhang Long , Gou Zhenjiu , Li Shunpeng , Freilich Shiri , Jiang Jiandong 
Published: December 2015

ABSTRACTComamonasplasmids play important roles in shaping the phenotypes of their hosts and the adaptation of these hosts to changing environments, and understanding the evolutionary strategy of these plasmids is thus of great concern. In this study, the sequence of the 119-kb 3,5-dibromo-4-hydroxybenzonitrile-catabolizing plasmid pBHB fromComamonassp. strain 7D-2 was studied and compared with those of three otherComamonashaloaromatic catabolic plasmids. Incompatibility group determination based on a phylogenetic analysis of 24 backbone gene proteins, as well as TrfA, revealed that these four plasmids all belong to the IncP-1β subgroup. Comparison of the four plasmids revealed a conserved backbone region and diverse genetic-load regions. The four plasmids share a core genome consisting of 40 genes (>50% similarities) and contain 12 to 50 unique genes each, most of which are xenobiotic-catabolic genes. Two functional reductive dehalogenase gene clusters are specifically located on pBHB, showing distinctive evolution of pBHB for haloaromatics. The higher catabolic ability of thebhbA2B2cluster than thebhbABcluster may be due to the transcription levels and the character of the dehalogenase gene itself rather than that of its extracytoplasmic binding receptor gene. The plasmid pBHB is riddled with transposons and insertion sequence (IS) elements, and ISs play important roles in the evolution of pBHB. The analysis of the origin of thebhbgenes on pBHB suggested that these accessory genes evolved independently. Our work provides insights into the evolutionary strategies ofComamonasplasmids, especially into the adaptation mechanism employed by pBHB for haloaromatics.
Levan-Producing Leuconostoc citreum Strain BD1707 and Its Growth in Tomato Juice Supplemented with Sucrose
Authors: Han Jin , Xu Xiaofen , Gao Caixia , Liu Zhenmin , Wu Zhengjun 
Published: December 2015

ABSTRACT

A levan-producing strain, BD1707, was isolated from Tibetan kefir and identified as
Leuconostoc citreum
. The effects of carbon sources on the growth of
L. citreum
BD1707 and levan production in tomato juice were measured. The changes in pH, viable cell count, sugar content, and levan yield in the cultured tomato juice supplemented with 15% (wt/vol) sucrose were also assayed.
L. citreum
BD1707 could synthesize more than 28 g/liter of levan in the tomato juice-sucrose medium when cultured at 30°C for 96 h. Based on the monosaccharide composition, molecular mass distribution, Fourier transform infrared (FTIR) spectra, and nuclear magnetic resonance (NMR) spectra, the levan synthesized by
L. citreum
BD1707 was composed of a linear backbone consisting of consecutive β-(2→6) linked
d
-fructofuranosyl units, with an estimated average molecular mass of 4.3 × 10
6
Da.
Chloroflexi CL500-11 Populations That Predominate Deep-Lake Hypolimnion Bacterioplankton Rely on Nitrogen-Rich Dissolved Organic Matter Metabolism and C
1
Compound Oxidation
Authors: Denef Vincent J. , Mueller Ryan S. , Chiang Edna , Liebig James R. , Vanderploeg Henry A. 
Published: December 2015

ABSTRACT

The
Chloroflexi
CL500-11 clade contributes a large proportion of the bacterial biomass in the oxygenated hypolimnia of deep lakes worldwide, including the world's largest freshwater system, the Laurentian Great Lakes. Traits that allow CL500-11 to thrive and its biogeochemical role in these environments are currently unknown. Here, we found that a CL500-11 population was present mostly in offshore waters along a transect in ultraoligotrophic Lake Michigan (a Laurentian Great Lake). It occurred throughout the water column in spring and only in the hypolimnion during summer stratification, contributing up to 18.1% of all cells. Genome reconstruction from metagenomic data suggested an aerobic, motile, heterotrophic lifestyle, with additional energy being gained through carboxidovory and methylovory. Comparisons to other available streamlined freshwater genomes revealed that the CL500-11 genome contained a disproportionate number of cell wall/capsule biosynthesis genes and the most diverse spectrum of genes involved in the uptake of dissolved organic matter (DOM) substrates, particularly peptides.
In situ
expression patterns indicated the importance of DOM uptake and protein/peptide turnover, as well as type I and type II carbon monoxide dehydrogenase and flagellar motility. Its location in the water column influenced its gene expression patterns the most. We observed increased bacteriorhodopsin gene expression and a response to oxidative stress in surface waters compared to its response in deep waters. While CL500-11 carries multiple adaptations to an oligotrophic lifestyle, its investment in motility, its large cell size, and its distribution in both oligotrophic and mesotrophic lakes indicate its ability to thrive under conditions where resources are more plentiful. Our data indicate that CL500-11 plays an important role in nitrogen-rich DOM mineralization in the extensive deep-lake hypolimnion habitat.
Ectomycorrhizal Fungal Protein Degradation Ability Predicted by Soil Organic Nitrogen Availability
Authors: Rineau Francois , Stas Jelle , Nguyen Nhu H. , Kuyper Thomas W. , Carleer Robert , Vangronsveld Jaco , Colpaert Jan V. , Kennedy Peter G. 
Published: December 2015

ABSTRACT

In temperate and boreal forest ecosystems, nitrogen (N) limitation of tree metabolism is alleviated by ectomycorrhizal (ECM) fungi. As forest soils age, the primary source of N in soil switches from inorganic (NH
4
+
and NO
3
−
) to organic (mostly proteins). It has been hypothesized that ECM fungi adapt to the most common N source in their environment, which implies that fungi growing in older forests would have greater protein degradation abilities. Moreover, recent results for a model ECM fungal species suggest that organic N uptake requires a glucose supply. To test the generality of these hypotheses, we screened 55 strains of 13
Suillus
species with different ecological preferences for their
in vitro
protein degradation abilities.
Suillus
species preferentially occurring in mature forests, where soil contains more organic matter, had significantly higher protease activity than those from young forests with low-organic-matter soils or species indifferent to forest age. Within species, the protease activities of ecotypes from soils with high or low soil organic N content did not differ significantly, suggesting resource partitioning between mineral and organic soil layers. The secreted protease mixtures were strongly dominated by aspartic peptidases. Glucose addition had variable effects on secreted protease activity; in some species, it triggered activity, but in others, activity was repressed at high concentrations. Collectively, our results indicate that protease activity, a key ectomycorrhizal functional trait, is positively related to environmental N source availability but is also influenced by additional factors, such as carbon availability.
Contribution of the Salmonella enterica KdgR Regulon to Persistence of the Pathogen in Vegetable Soft Rots
Authors: George Andrée S. , Salas González Isai , Lorca Graciela L. , Teplitski Max 
Published: December 2015

ABSTRACT

During their colonization of plants, human enteric pathogens, such as
Salmonella enterica
, are known to benefit from interactions with phytopathogens. At least in part, benefits derived by
Salmonella
from the association with a soft rot caused by
Pectobacterium carotovorum
were shown to be dependent on
Salmonella
KdgR, a regulator of genes involved in the uptake and utilization of carbon sources derived from the degradation of plant polymers. A
Salmonella
kdgR
mutant was more fit in soft rots but not in the lesions caused by
Xanthomonas
spp. and
Pseudomonas
spp. Bioinformatic, phenotypic, and gene expression analyses demonstrated that the KdgR regulon included genes involved in uptake and metabolism of molecules resulting from pectin degradation as well as those central to the utilization of a number of other carbon sources. Mutant analyses indicated that the Entner-Doudoroff pathway, in part controlled by KdgR, was critical for the persistence within soft rots and likely was responsible for the
kdgR
phenotype.
Chirality Matters: Synthesis and Consumption of the
d
-Enantiomer of Lactic Acid by Synechocystis sp. Strain PCC6803
Authors: Angermayr S. Andreas , van der Woude Aniek D. , Correddu Danilo , Kern Ramona , Hagemann Martin , Hellingwerf Klaas J. 
Published: December 2015

ABSTRACT

Both enantiomers of lactic acid,
l
-lactic acid and
d
-lactic acid, can be produced in a sustainable way by a photosynthetic microbial cell factory and thus from CO
2
, sunlight, and water. Several properties of polylactic acid (a polyester of polymerized lactic acid) depend on the controlled blend of these two enantiomers. Recently, cyanobacterium
Synechocystis
sp. strain PCC6803 was genetically modified to allow formation of either of these two enantiomers. This report elaborates on the
d
-lactic acid production achieved by the introduction of a
d
-specific lactate dehydrogenase from the lactic acid bacterium
Leuconostoc mesenteroides
into
Synechocystis
. A typical batch culture of this recombinant strain initially shows lactic acid production, followed by a phase of lactic acid consumption, until production “outcompetes” consumption at later growth stages. We show that
Synechocystis
is able to use
d
-lactic acid, but not
l
-lactic acid, as a carbon source for growth. Deletion of the organism's putative
d
-lactate dehydrogenase (encoded by
slr1556
), however, does not eliminate this ability with respect to
d
-lactic acid consumption. In contrast,
d
-lactic acid consumption does depend on the presence of glycolate dehydrogenase GlcD1 (encoded by
sll0404
). Accordingly, this report highlights the need to match a product of interest of a cyanobacterial cell factory with the metabolic network present in the host used for its synthesis and emphasizes the need to understand the physiology of the production host in detail.
Activating and Elucidating Metabolism of Complex Sugars in Yarrowia lipolytica
Authors: Ryu Seunghyun , Hipp Julie , Trinh Cong T. 
Published: December 2015

ABSTRACT

The oleaginous yeast
Yarrowia lipolytica
is an industrially important host for production of organic acids, oleochemicals, lipids, and proteins with broad biotechnological applications. Albeit known for decades, the unique native metabolism of
Y. lipolytica
for using complex fermentable sugars, which are abundant in lignocellulosic biomass, is poorly understood. In this study, we activated and elucidated the native sugar metabolism in
Y. lipolytica
for cell growth on xylose and cellobiose as well as their mixtures with glucose through comprehensive metabolic and transcriptomic analyses. We identified 7 putative glucose-specific transporters, 16 putative xylose-specific transporters, and 4 putative cellobiose-specific transporters that are transcriptionally upregulated for growth on respective single sugars.
Y. lipolytica
is capable of using xylose as a carbon source, but xylose dehydrogenase is the key bottleneck of xylose assimilation and is transcriptionally repressed by glucose.
Y. lipolytica
has a set of 5 extracellular and 6 intracellular β-glucosidases and is capable of assimilating cellobiose via extra- and intracellular mechanisms, the latter being dominant for growth on cellobiose as a sole carbon source. Strikingly,
Y. lipolytica
exhibited enhanced sugar utilization for growth in mixed sugars, with strong carbon catabolite activation for growth on the mixture of xylose and cellobiose and with mild carbon catabolite repression of glucose on xylose and cellobiose. The results of this study shed light on fundamental understanding of the complex native sugar metabolism of
Y. lipolytica
and will help guide inverse metabolic engineering of
Y. lipolytica
for enhanced conversion of biomass-derived fermentable sugars to chemicals and fuels.
Identification of Unknown Carboxydovore Bacteria Dominant in Deciduous Forest Soil via Succession of Bacterial Communities,
coxL
Genotypes, and Carbon Monoxide Oxidation Activity in Soil Microcosms
Authors: Lalonde Isabelle , Constant Philippe 
Published: December 2015

ABSTRACT

Surveys of the
coxL
gene, encoding the large subunit of the CO dehydrogenase, are used as a standard approach in ecological studies of carboxydovore bacteria scavenging atmospheric CO. Recent soil surveys unveiled that the distribution of
coxL
sequences encompassing the atypical genotype
coxL
type I group x was correlated to the CO oxidation activity. Based on phylogenetic analysis including the available
coxL
reference genome sequences, this unusual genotype was assigned to an unknown member of the
Deltaproteobacteria
, with the
coxL
sequence from
Haliangium ochraceum
being the sole and closest reference sequence. Here we seek to challenge the proposed taxonomic assignation of the
coxL
group x genotype through the monitoring of CO consumption activity and microbial community successions during the colonization of sterile soil microcosms inoculated with indigenous microorganisms. In our study, we established that the estimated population density of
Deltaproteobacteria
was too small to account for the abundance of the
coxL
group x genotype detected in soil. Furthermore, we computed a correlation network to relate 16S rRNA gene profiles with the succession of
coxL
genotypes and CO uptake activity in soil. We found that most of the
coxL
genotypes for which the colonization profile displayed covariance with CO uptake activity were related to potential carboxydovore bacteria belonging to
Actinobacteria
and
Alphaproteobacteria
. Our analysis did not provide any evidence that
coxL
group x genotypes belonged to
Deltaproteobacteria
. Considering the colonization profile of CO-oxidizing bacteria and the theoretical energy yield of measured CO oxidation rates in soil microcosms, we propose that unknown carboxydovore bacteria harboring the atypical
coxL
group x genotype are mixotrophic
K
-strategists.
Respiratory Kinetics of Marine Bacteria Exposed to Decreasing Oxygen Concentrations
Authors: Gong Xianzhe , Garcia-Robledo Emilio , Schramm Andreas , Revsbech Niels Peter 
Published: December 2015

ABSTRACTDuring aerobic respiration, microorganisms consume oxygen (O2) through the use of different types of terminal oxidases which have a wide range of affinities for O2. TheKmvalues for O2of these enzymes have been determined to be in the range of 3 to 200 nmol liter−1. In this study, we examined the time course of development of aerobic respiratory kinetics of four marine bacterial species (Dinoroseobacter shibae,Roseobacter denitrificans,Idiomarina loihiensis, andMarinobacter daepoensis) during exposure to decreasing O2concentrations. The genomes of all four species have genes for both high-affinity and low-affinity terminal oxidases. The respiration rate of the bacteria was measured by the use of extremely sensitive optical trace O2sensors (range, 1 to 1,000 nmol liter−1). Three of the four isolates exhibited apparentKmvalues of 30 to 60 nmol liter−1when exposed to submicromolar O2concentrations, but a decrease to values below 10 nmol liter−1was observed when the respiration rate per cell was lowered and the cell size was decreased due to starvation. The fourth isolate did not reach a low respiration rate per cell during starvation and exhibited apparentKmvalues of about 20 nmol liter−1throughout the experiment. The results clearly demonstrate not only that enzyme kinetics may limit O2uptake but also that even individual cells may be diffusion limited and that this diffusion limitation is the most pronounced at high respiration rates. A decrease in cell size by starvation, due to limiting organic carbon, and thereby more efficient diffusion uptake may also contribute to lower apparentKmvalues.
pyrF
as a Counterselectable Marker for Unmarked Genetic Manipulations in Treponema denticola
Authors: Kurniyati Kurni , Li Chunhao 
Published: December 2015

ABSTRACT

The pathophysiology of
Treponema denticola
, an oral pathogen associated with both periodontal and endodontic infections, is poorly understood due to its fastidious growth and recalcitrance to genetic manipulations. Counterselectable markers are instrumental in constructing clean and unmarked mutations in bacteria. Here, we demonstrate that
pyrF
, a gene encoding orotidine-5′-monophosphate decarboxylase, can be used as a counterselectable marker in
T. denticola
to construct marker-free mutants.
T. denticola
is susceptible to 5-fluoroorotic acid (5-FOA). To establish a
pyrF
-based counterselectable knockout system in
T. denticola
, the
pyrF
gene was deleted. The deletion conferred resistance to 5-FOA in
T. denticola
. Next, a single-crossover mutant was constructed by reintroducing
pyrF
along with a gentamicin resistance gene (
aacC1
) back into the chromosome of the
pyrF
mutant at the locus of choice. In this study, we chose
flgE
, a flagellar hook gene that is located within a large polycistronic motility gene operon, as our target gene. The obtained single-crossover mutant (named FlgE
in
) regained the susceptibility to 5-FOA. Finally, FlgE
in
was plated on solid agar containing 5-FOA. Numerous colonies of the 5-FOA-resistant mutant (named FlgE
out
) were obtained and characterized by PCR and Southern blotting analyses. The results showed that the
flgE
gene was deleted and FlgE
out
was free of selection markers (i.e.,
pyrF
and
aacC1
). Compared to previously constructed
flgE
mutants that contain an antibiotic selection marker, the deletion of
flgE
in FlgE
out
has no polar effect on its downstream gene expression. The system developed here will provide us with a new tool for investigating the genetics and pathogenicity of
T. denticola
.
Distributions of Fecal Markers in Wastewater from Different Climatic Zones for Human Fecal Pollution Tracking in Australian Surface Waters
Authors: Ahmed W. , Sidhu J. P. S. , Smith K. , Beale D. J. , Gyawali P. , Toze S. 
Published: December 2015

ABSTRACT

Recreational and potable water supplies polluted with human wastewater can pose a direct health risk to humans. Therefore, sensitive detection of human fecal pollution in environmental waters is very important to water quality authorities around the globe. Microbial source tracking (MST) utilizes human fecal markers (HFMs) to detect human wastewater pollution in environmental waters. The concentrations of these markers in raw wastewater are considered important because it is likely that a marker whose concentration is high in wastewater will be more frequently detected in polluted waters. In this study, quantitative PCR (qPCR) assays were used to determine the concentrations of fecal indicator bacteria (FIB)
Escherichia coli
and
Enterococcus
spp., HFMs
Bacteroides
HF183, human adenoviruses (HAdVs), and polyomaviruses (HPyVs) in raw municipal wastewater influent from various climatic zones in Australia.
E. coli
mean concentrations in pooled human wastewater data sets (from various climatic zones) were the highest (3.2 × 10
6
gene copies per ml), followed by those of HF183 (8.0 × 10
5
gene copies per ml) and
Enterococcus
spp. (3.6 × 10
5
gene copies per ml). HAdV and HPyV concentrations were 2 to 3 orders of magnitude lower than those of FIB and HF183. Strong positive and negative correlations were observed between the FIB and HFM concentrations within and across wastewater treatment plants (WWTPs). To identify the most sensitive marker of human fecal pollution, environmental water samples were seeded with raw human wastewater. The results from the seeding experiments indicated that
Bacteroides
HF183 was more sensitive for detecting human fecal pollution than HAdVs and HPyVs. Since the HF183 marker can occasionally be present in nontarget animal fecal samples, it is recommended that HF183 along with a viral marker (HAdVs or HPyVs) be used for tracking human fecal pollution in Australian environmental waters.
The impact of short periods of match congestion on injury risk and patterns in an elite football club
Authors: Carling Chris , McCall Alan , Le Gall Franck , Dupont Gregory 
Published: December 2015

BackgroundThe effect of fixture congestion on injury rates and patterns has received scarce attention in elite football and existing investigations have not accounted for player rotation or examined the temporal distribution and potential cause of injuries.AimTo prospectively investigate the epidemiology of injury during short periods of fixture congestion in a professional football club.MethodsOver a six-season period, exposure time and injury data were compared in the same players (n=25 (14 individuals)) when participating in two frequently occurring short congested fixture cycles in comparison to match-play outside these cycles. (1) two successive matches separated by an interval totalling ≤3 days calculated immediately from the end of play in match 1 to the beginning of play in match 2; (2) three successive matches separated by ≤4-day intervals starting the day immediately after each match.ResultsIn two-match congestion cycles, incidence rate ratios (IRR) showed that there was a higher risk of injury in the final 15 min of play in the second match in comparison to match-play outside the cycles (IRR: 3.1 (95% CI 1.1 to 9.3), p=0.0400). A greater risk of injury overall (IRR: 2.0 (95% CI 1.1 to 3.8), p=0.0345) and in the first-half of play (2.6 (1.1 to 6,5), p=0.0386), and risk of ankle sprains (10.4 (95% CI 1.9 to 57.9), p=0.0068) and non-contact injuries due to a ‘change in direction’ (IRR: 7.8 (1.3 to 46.8), p=0.0243) were observed in the final match of three-match congestion cycles in comparison to match-play outside the cycles.ConclusionsInjury rates and patterns were affected in the same elite football players when competing in short congested fixture cycles in comparison to match-play outside the cycles.