bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform
Authors: Kozich James J. , Westcott Sarah L. , Baxter Nielson T. , Highlander Sarah K. , Schloss Patrick D. 
Published: June 2013

ABSTRACT
Rapid advances in sequencing technology have changed the experimental landscape of microbial ecology. In the last 10 years, the field has moved from sequencing hundreds of 16S rRNA gene fragments per study using clone libraries to the sequencing of millions of fragments per study using next-generation sequencing technologies from 454 and Illumina. As these technologies advance, it is critical to assess the strengths, weaknesses, and overall suitability of these platforms for the interrogation of microbial communities. Here, we present an improved method for sequencing variable regions within the 16S rRNA gene using Illumina's MiSeq platform, which is currently capable of producing paired 250-nucleotide reads. We evaluated three overlapping regions of the 16S rRNA gene that vary in length (i.e., V34, V4, and V45) by resequencing a mock community and natural samples from human feces, mouse feces, and soil. By titrating the concentration of 16S rRNA gene amplicons applied to the flow cell and using a quality score-based approach to correct discrepancies between reads used to construct contigs, we were able to reduce error rates by as much as two orders of magnitude. Finally, we reprocessed samples from a previous study to demonstrate that large numbers of samples could be multiplexed and sequenced in parallel with shotgun metagenomes. These analyses demonstrate that our approach can provide data that are at least as good as that generated by the 454 platform while providing considerably higher sequencing coverage for a fraction of the cost.
Increases in the Amounts of Vibrio spp. in Oysters upon Addition of Exogenous Bacteria
Authors: Froelich Brett , Oliver James 
Published: June 2013

ABSTRACT

The bacterial pathogen
Vibrio vulnificus
is found naturally in brackish coastal waters but can be greatly concentrated by filter-feeding organisms such as shellfish. Numerous experiments in which exogenous
V. vulnificus
cells are added to oysters in an attempt to measure uptake and depuration have been performed. In nearly all cases, results have shown that laboratory-grown bacteria are rapidly taken up by the oysters but ultimately eliminated, while naturally present
Vibrio
populations in oysters are resistant to depuration. In this study, oysters harvested during winter months, with low culturable
Vibrio
concentrations, were incubated in aquaria supplemented with strains of
V. vulnificus
that were either genotypically or phenotypically distinct from the background bacteria. These exogenous cells were eliminated from the oysters, as previously seen, but other vibrios already inhabiting the oysters responded to the
V. vulnificus
inoculum by rapidly increasing in number and maintaining a large stable population. The presence of such an oyster-adapted
Vibrio
population would be expected to prevent colonization by exogenous
V. vulnificus
cells, thus explaining the rapid depuration of these added bacteria.
Relationship between Presence of Cows with Milk Positive for Mycobacterium avium subsp. paratuberculosis-Specific Antibody by Enzyme-Linked Immunosorbent Assay and Viable M. avium subsp. paratuberculosis in Dust in Cattle Barns
Authors: Eisenberg Susanne W. F. , Chuchaisangrat Ruj , Nielen Mirjam , Koets Ad P. 
Published: June 2013

ABSTRACTParatuberculosis, or Johne's disease, in cattle is caused byMycobacterium aviumsubsp.paratuberculosis, which has recently been suspected to be transmitted through dust. This longitudinal study on eight commercialM. aviumsubsp.paratuberculosis-positive dairy farms studied the relationship between the number of cows withM. aviumsubsp.paratuberculosisantibody-positive milk and the presence of viableM. aviumsubsp.paratuberculosisin settled-dust samples, including their temporal relationship. Milk and dust samples were collected in parallel monthly for 2 years.M. aviumsubsp.paratuberculosisantibodies in milk were measured by enzyme-linked immunosorbent assay (ELISA) and used as a proxy forM. aviumsubsp.paratuberculosisshedding. Settled-dust samples were collected by using electrostatic dust collectors (EDCs) at six locations in housing for dairy cattle and young stock. The presence of viableM. aviumsubsp.paratuberculosiswas identified by liquid culture and PCR. The results showed a positive relationship (odds ratio [OR], 1.2) between the number of cows with ELISA-positive milk and the odds of having positive EDCs in the same airspace as the adult dairy cattle. Moreover, the total number of lactating cows also showed an OR slightly above 1. This relationship remained the same for settled-dust samples collected up to 2 months before or after the time of milk sampling. The results suggest that removal of adult cows with milk positive forM. aviumsubsp.paratuberculosis-specific antibody by ELISA might result in a decrease in the presence of viableM. aviumsubsp.paratuberculosisin dust and therefore in the environment. However, this decrease is likely delayed by several weeks at least. In addition, the data support the notion thatM. aviumsubsp.paratuberculosisexposure of young stock is reduced by separate housing.
Saccharification of Cellulose by Recombinant Rhodococcus opacus PD630 Strains
Authors: Hetzler Stephan , Bröker Daniel , Steinbüchel Alexander 
Published: June 2013

ABSTRACT

The noncellulolytic actinomycete
Rhodococcus opacus
strain PD630 is the model oleaginous prokaryote with regard to the accumulation and biosynthesis of lipids, which serve as carbon and energy storage compounds and can account for as much as 87% of the dry mass of the cell in this strain. In order to establish cellulose degradation in
R. opacus
PD630, we engineered strains that episomally expressed six different cellulase genes from
Cellulomonas fimi
ATCC 484 (
cenABC
,
cex
,
cbhA
) and
Thermobifida fusca
DSM43792 (
cel6A
), thereby enabling
R. opacus
PD630 to degrade cellulosic substrates to cellobiose. Of all the enzymes tested, five exhibited a cellulase activity toward carboxymethyl cellulose (CMC) and/or microcrystalline cellulose (MCC) as high as 0.313 ± 0.01 U · ml
−1
, but recombinant strains also hydrolyzed cotton, birch cellulose, copy paper, and wheat straw. Cocultivations of recombinant strains expressing different cellulase genes with MCC as the substrate were carried out to identify an appropriate set of cellulases for efficient hydrolysis of cellulose by
R. opacus
. Based on these experiments, the multicellulase gene expression plasmid pCellulose was constructed, which enabled
R. opacus
PD630 to hydrolyze as much as 9.3% ± 0.6% (wt/vol) of the cellulose provided. For the direct production of lipids from birch cellulose, a two-step cocultivation experiment was carried out. In the first step, 20% (wt/vol) of the substrate was hydrolyzed by recombinant strains expressing the whole set of cellulase genes. The second step was performed by a recombinant cellobiose-utilizing strain of
R. opacus
PD630, which accumulated 15.1% (wt/wt) fatty acids from the cellobiose formed in the first step.
Serotypes, Virulence Factors, and Antimicrobial Susceptibilities of Vaginal and Fecal Isolates of Escherichia coli from Giant Pandas
Authors: Wang Xin , Yan Qigui , Xia Xiaodong , Zhang Yanming , Li Desheng , Wang Chengdong , Chen Shijie , Hou Rong 
Published: June 2013

ABSTRACT

Although
Escherichia coli
typically colonizes the intestinal tract and vagina of giant pandas, it has caused enteric and systemic disease in giant pandas and greatly impacts the health and survival of this endangered species. In order to understand the distribution and characteristics of
E. coli
from giant pandas, 67 fecal and 30 vaginal
E. coli
isolates from 21 giant pandas were characterized for O serogroups, phylogenetic groups, antimicrobial susceptibilities, and pulsed-field gel electrophoresis (PFGE) profiles. In addition, these isolates were tested for the presence of extraintestinal pathogenic
E. coli
(ExPEC) and diarrheagenic
E. coli
(DEC) by multiplex PCR detection of specific virulence genes. The most prevalent serogroups for all
E. coli
isolates were O88, O18, O167, O4, and O158. ExPEC isolates were detected mostly in vaginal samples, and DEC isolates were detected only in fecal samples. Phylogenetic group B1 predominated in fecal isolates, while groups B2 and D were frequently detected in vaginal isolates. Resistance to trimethoprim-sulfamethoxazole was most frequently observed, followed by resistance to nalidixic acid and tetracycline. All except five isolates were typeable by using XbaI and were categorized into 74 PFGE patterns. Our findings indicate that panda
E. coli
isolates exhibited antimicrobial resistance, and potentially pathogenic
E. coli
isolates were present in giant pandas. In addition, these
E. coli
isolates were genetically diverse. This study may provide helpful information for developing strategies in the future to control
E. coli
infections of giant pandas.
Haloarchaeal-Type β-Ketothiolases Involved in Poly(3-Hydroxybutyrate-
co
-3-Hydroxyvalerate) Synthesis in Haloferax mediterranei
Authors: Hou Jing , Feng Bo , Han Jing , Liu Hailong , Zhao Dahe , Zhou Jian , Xiang Hua 
Published: June 2013

ABSTRACT

The key enzymes for poly(3-hydroxybutyrate-
co
-3-hydroxyvalerate) (PHBV) biosynthesis in haloarchaea have been identified except the β-ketothiolase(s), which condense two acetyl coenzyme A (acetyl-CoA) molecules to acetoacetyl-CoA, or one acetyl-CoA and one propionyl-CoA to 3-ketovaleryl-CoA. Whole-genome analysis has revealed eight potential β-ketothiolase genes in the haloarchaeon
Haloferax mediterranei
, among which the PHBV-specific BktB and PhaA were identified by gene knockout and complementation analysis. Unlike all known bacterial counterparts encoded by a single gene, the haloarchaeal PhaA that was involved in acetoacetyl-CoA generation, was composed of two different types of subunits (PhaAα and PhaAβ) and encoded by the cotranscribed HFX_1023 (
phaA
α) and HFX_1022 (
phaA
β) genes. Similarly, the BktB that was involved in generation of acetoacetyl-CoA and 3-ketovaleryl-CoA, was also composed of two different types of subunits (BktBα and BktBβ) and encoded by cotranscribed HFX_6004 (
bktB
α) and HFX_6003 (
bktB
β). BktBα and PhaAα were the catalytic subunits and determined substrate specificities of BktB and PhaA, respectively. Their catalytic triad “Ser-His-His” was distinct from the bacterial “Cys-His-Cys.” BktBβ and PhaAβ both contained an oligosaccharide-binding fold domain, which was essential for the β-ketothiolase activity. Interestingly, BktBβ and PhaAβ were functionally interchangeable, although PhaAβ preferred functioning with PhaAα. In addition, BktB showed biotechnological potential for the production of PHBV with the desired 3-hydroxyvalerate fraction in haloarchaea. This is the first report of the haloarchaeal type of PHBV-specific β-ketothiolases, which are distinct from their bacterial counterparts in both subunit composition and catalytic residues.
Structure-Activity Relationship of Synthetic Variants of the Milk-Derived Antimicrobial Peptide α
s2
-Casein f(183–207)
Authors: Alvarez-Ordóñez Avelino , Begley Máire , Clifford Tanya , Deasy Thérèse , Considine Kiera , Hill Colin 
Published: June 2013

ABSTRACT

Template-based studies on antimicrobial peptide (AMP) derivatives obtained through manipulation of the amino acid sequence are helpful to identify properties or residues that are important for biological activity. The present study sheds light on the importance of specific amino acids of the milk-derived α
s2
-casein f(183–207) peptide to its antibacterial activity against the food-borne pathogens
Listeria monocytogenes
and
Cronobacter sakazakii
. Trimming of the peptide revealed that residues at the C-terminal end of the peptide are important for activity. Removal of the last 5 amino acids at the C-terminal end and replacement of the Arg at position 23 of the peptide sequence by an Ala residue significantly decreased activity. These findings suggest that Arg23 is very important for optimal activity of the peptide. Substitution of the also positively charged Lys residues at positions 15 and 17 of the α
s2
-casein f(183–207) peptide also caused a significant reduction of the effectiveness against
C. sakazakii
, which points toward the importance of the positive charge of the peptide for its biological activity. Indeed, simultaneous replacement of various positively charged amino acids was linked to a loss of bactericidal activity. On the other hand, replacement of Pro residues at positions 14 and 20 resulted in a significantly increased antibacterial potency, and hydrophobic end tagging of α
s2
-casein f(193–203) and α
s2
-casein f(197–207) peptides with multiple Trp or Phe residues significantly increased their potency against
L. monocytogenes
. Finally, the effect of pH (4.5 to 7.4), temperature (4°C to 37°C), and addition of sodium and calcium salts (1% to 3%) on the activity of the 15-amino-acid α
s2
-casein f(193–207) peptide was also determined, and its biological activity was shown to be completely abolished in high-saline environments.
Longitudinal Study of Distributions of Similar Antimicrobial-Resistant Salmonella Serovars in Pigs and Their Environment in Two Distinct Swine Production Systems
Authors: Keelara Shivaramu , Scott H. Morgan , Morrow William M. , Gebreyes Wondwossen A. , Correa Maria , Nayak Rajesh , Stefanova Rossina , Thakur Siddhartha 
Published: June 2013

ABSTRACTThe aim of this longitudinal study was to determine and compare the prevalences and genotypic profiles of antimicrobial-resistant (AR)Salmonellaisolates from pigs reared in antimicrobial-free (ABF) and conventional production systems at farm, at slaughter, and in their environment. We collected 2,889 pig fecal and 2,122 environmental (feed, water, soil, lagoon, truck, and floor swabs) samples from 10 conventional and eight ABF longitudinal cohorts at different stages of production (farrowing, nursery, finishing) and slaughter (postevisceration, postchill, and mesenteric lymph nodes [MLN]). In addition, we collected 1,363 carcass swabs and 205 lairage and truck samples at slaughter. A total of 1,090Salmonellaisolates were recovered from the samples; these were isolated with a significantly higher prevalence in conventionally reared pigs (4.0%;n= 66) and their environment (11.7%;n= 156) than in ABF pigs (0.2%;n= 2) and their environment (0.6%;n= 5) (P< 0.001).Salmonellawas isolated from all stages at slaughter, including the postchill step, in the two production systems.Salmonellaprevalence was significantly higher in MLN extracted from conventional carcasses than those extracted from ABF carcasses (P< 0.001). We identified a total of 24 different serotypes, withSalmonella entericaserovar Typhimurium,Salmonella entericaserovar Anatum,Salmonella entericaserovar Infantis, andSalmonella entericaserovar Derby being predominant. The highest frequencies of antimicrobial resistance (AR) were exhibited to tetracycline (71%), sulfisoxazole (42%), and streptomycin (17%). Multidrug resistance (resistance to ≥3 antimicrobials; MDR) was detected in 27% (n= 254) of theSalmonellaisolates from the conventional system. Our study reports a low prevalence ofSalmonellain both production systems in pigs on farms, while a higher prevalence was detected among the carcasses at slaughter. The dynamics ofSalmonellaprevalence in pigs and carcasses were reciprocated in the farm and slaughter environment, clearly indicating an exchange of this pathogen between the pigs and their surroundings. Furthermore, the phenotypic and genotypic fingerprint profile results underscore the potential role played by environmental factors in dissemination of ARSalmonellato pigs.
Polysaccharide-Degrading Thermophiles Generated by Heterologous Gene Expression in Geobacillus kaustophilus HTA426
Authors: Suzuki Hirokazu , Yoshida Ken-ichi , Ohshima Toshihisa 
Published: June 2013

ABSTRACTThermophiles have important advantages over mesophiles as host organisms for high-temperature bioprocesses, functional production of thermostable enzymes, and efficient expression of enzymatic activitiesin vivo. To capitalize on these advantages of thermophiles, we describe here a new inducible gene expression system in the thermophileGeobacillus kaustophilusHTA426. Six promoter regions in the HTA426 genome were identified and analyzed for expression profiles using β-galactosidase reporter assay. This analysis identified a promoter region upstream of a putative amylose-metabolizing gene cluster that directed high-level expression of the reporter gene. The expression was >280-fold that without a promoter and was further enhanced 12-fold by maltose addition. In association with a multicopy plasmid, this promoter region was used to express heterologous genes. Several genes, including a gene whose product was insoluble when expressed inEscherichia coli, were successfully expressed as soluble proteins, with yields of 0.16 to 59 mg/liter, and conferred new functions toG. kaustophilusstrains. Remarkably, cellulase and α-amylase genes conferred the ability to degrade cellulose paper and insoluble starch at high temperatures, respectively, generating thermophiles with the potential to degrade plant biomass. Our results demonstrate that this novel expression system expands the potential applications ofG. kaustophilus.
Characterization of a Feruloyl Esterase from Lactobacillus plantarum
Authors: Esteban-Torres María , Reverón Inés , Mancheño José Miguel , de las Rivas Blanca , Muñoz Rosario 
Published: June 2013

ABSTRACT

Lactobacillus plantarum
is frequently found in the fermentation of plant-derived food products, where hydroxycinnamoyl esters are abundant.
L. plantarum
WCFS1 cultures were unable to hydrolyze hydroxycinnamoyl esters; however, cell extracts from the strain partially hydrolyze methyl ferulate and methyl
p
-coumarate. In order to discover whether the protein Lp_0796 is the enzyme responsible for this hydrolytic activity, it was recombinantly overproduced and enzymatically characterized. Lp_0796 is an esterase that, among other substrates, is able to efficiently hydrolyze the four model substrates for feruloyl esterases (methyl ferulate, methyl caffeate, methyl
p
-coumarate, and methyl sinapinate). A screening test for the detection of the gene encoding feruloyl esterase Lp_0796 revealed that it is generally present among
L. plantarum
strains. The present study constitutes the description of feruloyl esterase activity in
L. plantarum
and provides new insights into the metabolism of hydroxycinnamic compounds in this bacterial species.
Novel, Oxygen-Insensitive Group 5 [NiFe]-Hydrogenase in Ralstonia eutropha
Authors: Schäfer Caspar , Friedrich Bärbel , Lenz Oliver 
Published: June 2013

ABSTRACT

Recently, a novel group of [NiFe]-hydrogenases has been defined that appear to have a great impact in the global hydrogen cycle. This so-called group 5 [NiFe]-hydrogenase is widespread in soil-living actinobacteria and can oxidize molecular hydrogen at atmospheric levels, which suggests a high affinity of the enzyme toward H
2
. Here, we provide a biochemical characterization of a group 5 hydrogenase from the betaproteobacterium
Ralstonia eutropha
H16. The hydrogenase was designated an actinobacterial hydrogenase (AH) and is catalytically active, as shown by the
in vivo
H
2
uptake and by activity staining in native gels. However, the enzyme does not sustain autotrophic growth on H
2
. The AH was purified to homogeneity by affinity chromatography and consists of two subunits with molecular masses of 65 and 37 kDa. Among the electron acceptors tested, nitroblue tetrazolium chloride was reduced by the AH at highest rates. At 30°C and pH 8, the specific activity of the enzyme was 0.3 μmol of H
2
per min and mg of protein. However, an unexpectedly high Michaelis constant (

K
m

) for H
2
of 3.6 ± 0.5 μM was determined, which is in contrast to the previously proposed low

K
m

of group 5 hydrogenases and makes atmospheric H
2
uptake by
R. eutropha
most unlikely. Amperometric activity measurements revealed that the AH maintains full H
2
oxidation activity even at atmospheric oxygen concentrations, showing that the enzyme is insensitive toward O
2
.
Exploring Environmental Control of Cyclic di-GMP Signaling in Vibrio cholerae by Using theEx VivoLysate Cyclic di-GMP Assay (TELCA)
Authors: Koestler Benjamin J. , Waters Christopher M. 
Published: June 2013

ABSTRACTVibrio choleraesenses its environment, including the surrounding bacterial community, using both the second messenger cyclic di-GMP (c-di-GMP) and quorum sensing (QS) to regulate biofilm formation and other bacterial behaviors. Cyclic di-GMP is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes.V. choleraeencodes a complex network of 61 enzymes predicted to mediate changes to the levels of c-di-GMP in response to extracellular signals, and the transcription of many of these enzymes is influenced by QS. Because of the complexity of the c-di-GMP signaling system inV. cholerae, it is difficult to determine if modulation of intracellular c-di-GMP in response to different stimuli is driven primarily by changes in c-di-GMP synthesis or hydrolysis. Here, we describe a novel method, named theex vivolysate c-di-GMP assay (TELCA), that systematically measures total DGC and PDE cellular activity. We show thatV. choleraegrown in different environments exhibits significantly different intracellular levels of c-di-GMP, and we used TELCA to determine that these differences correspond to changes in both c-di-GMP synthesis and hydrolysis. Furthermore, we show that the increased concentration of c-di-GMP at low cell density is primarily due to increased DGC activity due to the DGC CdgA. Our findings highlight the idea that modulation of both total DGC and PDE activity alters the intracellular concentration of c-di-GMP, and we present a new method that is widely applicable to the systematic analysis of complex c-di-GMP signaling networks.
Tracking Down Biotransformation to the Genetic Level: Identification of a Highly Flexible Glycosyltransferase from Saccharothrix espanaensis
Authors: Strobel Tina , Schmidt Yvonne , Linnenbrink Anton , Luzhetskyy Andriy , Luzhetska Marta , Taguchi Takaaki , Brötz Elke , Paululat Thomas , Stasevych Maryna , Stanko Oleg , Novikov Volodymyr , Bechthold Andreas 
Published: June 2013

ABSTRACT

Saccharothrix espanaensis
is a member of the order
Actinomycetales
. The genome of the strain has been sequenced recently, revealing 106 glycosyltransferase genes. In this paper, we report the detection of a glycosyltransferase from
Saccharothrix espanaensis
which is able to rhamnosylate different phenolic compounds targeting different positions of the molecules. The gene encoding the flexible glycosyltransferase is not located close to a natural product biosynthetic gene cluster. Therefore, the native function of this enzyme might be not the biosynthesis of a secondary metabolite but the glycosylation of internal and external natural products as part of a defense mechanism.