bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC 6803
Authors: Ng Andrew H. , Berla Bertram M. , Pakrasi Himadri B. 
Published: July 2015

ABSTRACT

Cyanobacteria are photosynthetic cell factories that use solar energy to convert CO
2
into useful products. Despite this attractive feature, the development of tools for engineering cyanobacterial chassis has lagged behind that for heterotrophs such as
Escherichia coli
or
Saccharomyces cerevisiae
. Heterologous genes in cyanobacteria are often integrated at presumptively “neutral” chromosomal sites, with unknown effects. We used transcriptome sequencing (RNA-seq) data for the model cyanobacterium
Synechocystis
sp. strain PCC 6803 to identify neutral sites from which no transcripts are expressed. We characterized the two largest such sites on the chromosome, a site on an endogenous plasmid, and a shuttle vector by integrating an enhanced yellow fluorescent protein (EYFP) expression cassette expressed from either the P
cpc560
or the P
trc1O
promoter into each locus. Expression from the endogenous plasmid was as much as 14-fold higher than that from the chromosome, with intermediate expression from the shuttle vector. The expression characteristics of each locus correlated predictably with the promoters used. These findings provide novel, characterized tools for synthetic biology and metabolic engineering in cyanobacteria.
Improvement in Thermostability of an Achaetomium sp. Strain Xz8 Endopolygalacturonase via the Optimization of Charge-Charge Interactions
Authors: Tu Tao , Luo Huiying , Meng Kun , Cheng Yanli , Ma Rui , Shi Pengjun , Huang Huoqing , Bai Yingguo , Wang Yaru , Zhang Lujia , Yao Bin 
Published: July 2015

ABSTRACT

Improving enzyme thermostability is of importance for widening the spectrum of application of enzymes. In this study, a structure-based rational design approach was used to improve the thermostability of a highly active, wide-pH-range-adaptable, and stable endopolygalacturonase (PG8fn) from
Achaetomium
sp. strain Xz8 via the optimization of charge-charge interactions. By using the enzyme thermal stability system (ETSS), two residues—D244 and D299—were inferred to be crucial contributors to thermostability. Single (D244A and D299R) and double (D244A/D299R) mutants were then generated and compared with the wild type. All mutants showed improved thermal properties, in the order D244A < D299R < D244A/D299R. In comparison with PG8fn, D244A/D299R showed the most pronounced shifts in temperature of maximum enzymatic activity (
T
max
), temperature at which 50% of the maximal activity of an enzyme is retained (
T
50
), and melting temperature (

T
m

), of about 10, 17, and 10.2°C upward, respectively, with the half-life (
t
1/2
) extended by 8.4 h at 50°C and 45 min at 55°C. Another distinguishing characteristic of the D244A/D299R mutant was its catalytic activity, which was comparable to that of the wild type (23,000 ± 130 U/mg versus 28,000 ± 293 U/mg); on the other hand, it showed more residual activity (8,400 ± 83 U/mg versus 1,400 ± 57 U/mg) after the feed pelleting process (80°C and 30 min). Molecular dynamics (MD) simulation studies indicated that mutations at sites D244 and D299 lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity. This study reveals the importance of charge-charge interactions in protein conformation and provides a viable strategy for enhancing protein stability.
Exposure of Salmonella enterica Serovar Typhimurium to Three Humectants Used in the Food Industry Induces Different Osmoadaptation Systems
Authors: Finn Sarah , Rogers Lisa , Händler Kristian , McClure Peter , Amézquita Alejandro , Hinton Jay C. D. , Fanning Séamus 
Published: July 2015

ABSTRACT

Common salt (NaCl) is frequently used by the food industry to add flavor and to act as a humectant in order to reduce the water content of a food product. The improved health awareness of consumers is leading to a demand for food products with reduced salt content; thus, manufacturers require alternative water activity-reducing agents which elicit the same general effects as NaCl. Two examples include KCl and glycerol. These agents lower the water activity of a food matrix and also contribute to limit the growth of the microbiota, including foodborne pathogens. Little is currently known about how foodborne pathogens respond to these water activity-lowering agents. Here we examined the response of
Salmonella enterica
serovar Typhimurium 4/74 to NaCl, KCl, and glycerol at three time points, using a constant water activity level, compared with the response of a control inoculum. All conditions induced the upregulation of gluconate metabolic genes after 6 h of exposure. Bacteria exposed to NaCl and KCl demonstrated the upregulation of the osmoprotective transporter mechanisms encoded by the
proP
,
proU
, and
osmU
(STM1491 to STM1494) genes. Glycerol exposure elicited the downregulation of these osmoadaptive mechanisms but stimulated an increase in lipopolysaccharide and membrane protein-associated genes after 1 h. The most extensive changes in gene expression occurred following exposure to KCl. Because many of these genes were of unknown function, further characterization may identify KCl-specific adaptive processes that are not stimulated by NaCl. This study shows that the response of
S
. Typhimurium to different humectants does not simply reflect reduced water activity and likely involves systems that are linked to specific humectants.
Novel Glucose-1-Phosphatase with High Phytase Activity and Unusual Metal Ion Activation from Soil Bacterium Pantoea sp. Strain 3.5.1
Authors: Suleimanova Aliya D. , Beinhauer Astrid , Valeeva Liia R. , Chastukhina Inna B. , Balaban Nelly P. , Shakirov Eugene V. , Greiner Ralf , Sharipova Margarita R. 
Published: July 2015

ABSTRACT

Phosphorus is an important macronutrient, but its availability in soil is limited. Many soil microorganisms improve the bioavailability of phosphate by releasing it from various organic compounds, including phytate. To investigate the diversity of phytate-hydrolyzing bacteria in soil, we sampled soils of various ecological habitats, including forest, private homesteads, large agricultural complexes, and urban landscapes. Bacterial isolate
Pantoea
sp. strain 3.5.1 with the highest level of phytase activity was isolated from forest soil and investigated further. The
Pantoea
sp. 3.5.1
agpP
gene encoding a novel glucose-1-phosphatase with high phytase activity was identified, and the corresponding protein was purified to apparent homogeneity, sequenced by mass spectroscopy, and biochemically characterized. The AgpP enzyme exhibits maximum activity and stability at pH 4.5 and at 37°C. The enzyme belongs to a group of histidine acid phosphatases and has the lowest

K
m

values toward phytate, glucose-6-phosphate, and glucose-1-phosphate. Unexpectedly, stimulation of enzymatic activity by several divalent metal ions was observed for the AgpP enzyme. High-performance liquid chromatography (HPLC) and high-performance ion chromatography (HPIC) analyses of phytate hydrolysis products identify
dl
-
myo
-inositol 1,2,4,5,6-pentakisphosphate as the final product of the reaction, indicating that the
Pantoea
sp. AgpP glucose-1-phosphatase can be classified as a 3-phytase. The identification of the
Pantoea
sp. AgpP phytase and its unusual regulation by metal ions highlight the remarkable diversity of phosphorus metabolism regulation in soil bacteria. Furthermore, our data indicate that natural forest soils harbor rich reservoirs of novel phytate-hydrolyzing enzymes with unique biochemical features.
Origin of the Outbreak in France of Pseudomonas syringae pv. actinidiae Biovar 3, the Causal Agent of Bacterial Canker of Kiwifruit, Revealed by a Multilocus Variable-Number Tandem-Repeat Analysis
Authors: Cunty A. , Cesbron S. , Poliakoff F. , Jacques M.-A. , Manceau C. 
Published: July 2015

ABSTRACTThe first outbreaks of bacterial canker of kiwifruit caused byPseudomonas syringaepv. actinidiae biovar 3 were detected in France in 2010.P. syringaepv. actinidiae causes leaf spots, dieback, and canker that sometimes lead to the death of the vine.P. syringaepv. actinidifoliorum, which is pathogenic on kiwi as well, causes only leaf spots. In order to conduct an epidemiological study to track the spread of the epidemics of these two pathogens in France, we developed a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA). MLVA was conducted on 340 strains ofP. syringaepv. actinidiae biovar 3 isolated in Chile, China, France, Italy, and New Zealand and on 39 strains ofP. syringaepv. actinidifoliorum isolated in Australia, France, and New Zealand. Eleven polymorphic VNTR loci were identified in the genomes ofP. syringaepv. actinidiae biovar 3 ICMP 18744 and ofP. syringaepv. actinidifoliorum ICMP 18807. MLVA enabled the structuring ofP. syringaepv. actinidiae biovar 3 andP. syringaepv. actinidifoliorum strains in 55 and 16 haplotypes, respectively. MLVA and discriminant analysis of principal components revealed that strains isolated in Chile, China, and New Zealand are genetically distinct fromP. syringaepv. actinidiae strains isolated in France and in Italy, which appear to be closely related at the genetic level. In contrast, no structuring was observed forP. syringaepv. actinidifoliorum. We developed an MLVA scheme to explore the diversity withinP. syringaepv. actinidiae biovar 3 and to trace the dispersal routes of epidemicP. syringaepv. actinidiae biovar 3 in Europe. We suggest using this MLVA scheme to trace the dispersal routes ofP. syringaepv. actinidiae at a global level.
The Bacterial Communities of Full-Scale Biologically Active, Granular Activated Carbon Filters Are Stable and Diverse and Potentially Contain Novel Ammonia-Oxidizing Microorganisms
Authors: LaPara Timothy M. , Hope Wilkinson Katheryn , Strait Jacqueline M. , Hozalski Raymond M. , Sadowksy Michael J. , Hamilton Matthew J. 
Published: July 2015

ABSTRACT

The bacterial community composition of the full-scale biologically active, granular activated carbon (BAC) filters operated at the St. Paul Regional Water Services (SPRWS) was investigated using Illumina MiSeq analysis of PCR-amplified 16S rRNA gene fragments. These bacterial communities were consistently diverse (Shannon index, >4.4; richness estimates, >1,500 unique operational taxonomic units [OTUs]) throughout the duration of the 12-month study period. In addition, only modest shifts in the quantities of individual bacterial populations were observed; of the 15 most prominent OTUs, the most highly variable population (a
Variovorax
sp.) modulated less than 13-fold over time and less than 8-fold from filter to filter. The most prominent population in the profiles was a
Nitrospira
sp., representing 13 to 21% of the community. Interestingly, very few of the known ammonia-oxidizing bacteria (AOB; <0.07%) and no ammonia-oxidizing
Archaea
were detected in the profiles. Quantitative PCR of
amoA
genes, however, suggested that AOB were prominent in the bacterial communities (
amoA
/16S rRNA gene ratio, 1 to 10%). We conclude, therefore, that the BAC filters at the SPRWS potentially contained significant numbers of unidentified and novel ammonia-oxidizing microorganisms that possess
amoA
genes similar to those of previously described AOB.
Distinct SagA from Hospital-Associated Clade A1 Enterococcus faecium Strains Contributes to Biofilm Formation
Authors: Paganelli F. L. , de Been M. , Braat J. C. , Hoogenboezem T. , Vink C. , Bayjanov J. , Rogers M. R. C. , Huebner J. , Bonten M. J. M. , Willems R. J. L. , Leavis H. L. 
Published: July 2015

ABSTRACT

Enterococcus faecium
is an important nosocomial pathogen causing biofilm-mediated infections. Elucidation of
E. faecium
biofilm pathogenesis is pivotal for the development of new strategies to treat these infections. In several bacteria, extracellular DNA (eDNA) and proteins act as matrix components contributing to biofilm development. In this study, we investigated biofilm formation capacity and the roles of eDNA and secreted proteins for 83
E. faecium
strains with different phylogenetic origins that clustered in clade A1 and clade B. Although there was no significant difference in biofilm formation between
E. faecium
strains from these two clades, the addition of DNase I or proteinase K to biofilms demonstrated that eDNA is essential for biofilm formation in most
E. faecium
strains, whereas proteolysis impacted primarily biofilms of
E. faecium
clade A1 strains. Secreted antigen A (SagA) was the most abundant protein in biofilms from
E. faecium
clade A1 and B strains, although its localization differed between the two groups.
sagA
was present in all sequenced
E. faecium
strains, with a consistent difference in the repeat region between the clades, which correlated with the susceptibility of biofilms to proteinase K. This indicates an association between the SagA variable repeat profile and the localization and contribution of SagA in
E. faecium
biofilms.
Transcriptomic Analysis of the Adaptation of Listeria monocytogenes to Growth on Vacuum-Packed Cold Smoked Salmon
Authors: Tang Silin , Orsi Renato H. , den Bakker Henk C. , Wiedmann Martin , Boor Kathryn J. , Bergholz Teresa M. 
Published: July 2015

ABSTRACT

The foodborne pathogen
Listeria monocytogenes
is able to survive and grow in ready-to-eat foods, in which it is likely to experience a number of environmental stresses due to refrigerated storage and the physicochemical properties of the food. Little is known about the specific molecular mechanisms underlying survival and growth of
L. monocytogenes
under different complex conditions on/in specific food matrices. Transcriptome sequencing (RNA-seq) was used to understand the transcriptional landscape of
L. monocytogenes
strain H7858 grown on cold smoked salmon (CSS; water phase salt, 4.65%; pH 6.1) relative to that in modified brain heart infusion broth (MBHIB; water phase salt, 4.65%; pH 6.1) at 7°C. Significant differential transcription of 149 genes was observed (false-discovery rate [FDR], <0.05; fold change, ≥2.5), and 88 and 61 genes were up- and downregulated, respectively, in H7858 grown on CSS relative to the genes in H7858 grown in MBHIB. In spite of these differences in transcriptomes under these two conditions, growth parameters for
L. monocytogenes
were not significantly different between CSS and MBHIB, indicating that the transcriptomic differences reflect how
L. monocytogenes
is able to facilitate growth under these different conditions. Differential expression analysis and Gene Ontology enrichment analysis indicated that genes encoding proteins involved in cobalamin biosynthesis as well as ethanolamine and 1,2-propanediol utilization have significantly higher transcript levels in H7858 grown on CSS than in that grown in MBHIB. Our data identify specific transcriptional profiles of
L. monocytogenes
growing on vacuum-packaged CSS, which may provide targets for the development of novel and improved strategies to control
L. monocytogenes
growth on this ready-to-eat food.
Characterization of
para
-Nitrophenol-Degrading Bacterial Communities in River Water by Using Functional Markers and Stable Isotope Probing
Authors: Kowalczyk Agnieszka , Eyice Özge , Schäfer Hendrik , Price Oliver R. , Finnegan Christopher J. , van Egmond Roger A. , Shaw Liz J. , Barrett Glyn , Bending Gary D. 
Published: July 2015

ABSTRACT

Microbial degradation is a major determinant of the fate of pollutants in the environment.
para
-Nitrophenol (PNP) is an EPA-listed priority pollutant with a wide environmental distribution, but little is known about the microorganisms that degrade it in the environment. We studied the diversity of active PNP-degrading bacterial populations in river water using a novel functional marker approach coupled with [
13
C
6
]PNP stable isotope probing (SIP). Culturing together with culture-independent terminal restriction fragment length polymorphism analysis of 16S rRNA gene amplicons identified
Pseudomonas syringae
to be the major driver of PNP degradation in river water microcosms. This was confirmed by SIP-pyrosequencing of amplified 16S rRNA. Similarly, functional gene analysis showed that degradation followed the Gram-negative bacterial pathway and involved
pnpA
from
Pseudomonas
spp. However, analysis of maleylacetate reductase (encoded by
mar
), an enzyme common to late stages of both Gram-negative and Gram-positive bacterial PNP degradation pathways, identified a diverse assemblage of bacteria associated with PNP degradation, suggesting that
mar
has limited use as a specific marker of PNP biodegradation. Both the
pnpA
and
mar
genes were detected in a PNP-degrading isolate,
P. syringae
AKHD2, which was isolated from river water. Our results suggest that PNP-degrading cultures of
Pseudomonas
spp. are representative of environmental PNP-degrading populations.
Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin
Authors: Lowrey Liam , Woodhams Douglas C. , Tacchi Luca , Salinas Irene 
Published: July 2015

ABSTRACT

The mucosal surfaces of wild and farmed aquatic vertebrates face the threat of many aquatic pathogens, including fungi. These surfaces are colonized by diverse symbiotic bacterial communities that may contribute to fight infection. Whereas the gut microbiome of teleosts has been extensively studied using pyrosequencing, this tool has rarely been employed to study the compositions of the bacterial communities present on other teleost mucosal surfaces. Here we provide a topographical map of the mucosal microbiome of an aquatic vertebrate, the rainbow trout (
Oncorhynchus mykiss
). Using 16S rRNA pyrosequencing, we revealed novel bacterial diversity at each of the five body sites sampled and showed that body site is a strong predictor of community composition. The skin exhibited the highest diversity, followed by the olfactory organ, gills, and gut.
Flectobacillus
was highly represented within skin and gill communities. Principal coordinate analysis and plots revealed clustering of external sites apart from internal sites. A highly diverse community was present within the epithelium, as demonstrated by confocal microscopy and pyrosequencing. Using
in vitro
assays, we demonstrated that two
Arthrobacter
sp. skin isolates, a
Psychrobacter
sp. strain, and a combined skin aerobic bacterial sample inhibit the growth of
Saprolegnia australis
and
Mucor hiemalis
, two important aquatic fungal pathogens. These results underscore the importance of symbiotic bacterial communities of fish and their potential role for the control of aquatic fungal diseases.
Effects of Chicken Litter Storage Time and Ammonia Content on Thermal Resistance of Desiccation-Adapted Salmonella spp
Authors: Chen Zhao , Wang Hongye , Ionita Claudia , Luo Feng , Jiang Xiuping 
Published: July 2015

ABSTRACT

Broiler chicken litter was kept as a stacked heap on a poultry farm, and samples were collected up to 9 months of storage. Chicken litter inoculated with desiccation-adapted
Salmonella
cells was heat-treated at 75, 80, 85, and 150°C.
Salmonella
populations decreased in all these samples during heat treatment, and the inactivation rates became lower in chicken litter when storage time was extended from 0 to 6 months. There was no significant difference (
P
> 0.05) in thermal resistance of
Salmonella
in 6- and 9-month litter samples, indicating that a threshold for thermal resistance was reached after 6 months. Overall, the thermal resistance of
Salmonella
in chicken litter was affected by the storage time of the litter. The changes in some chemical, physical, and microbiological properties during storage could possibly contribute to this difference. Moisture and ammonia could be two of the most significant factors influencing the thermal resistance of
Salmonella
cells in chicken litter. Our results emphasize the importance of adjusting time and temperature conditions for heat processing chicken litter when it is removed from the chicken house at different time intervals.
Aspergillus glaucus Aquaglyceroporin Gene
glpF
Confers High Osmosis Tolerance in Heterologous Organisms
Authors: Liu Xiao-Dan , Wei Yi , Zhou Xiao-Yang , Pei Xue , Zhang Shi-Hong 
Published: July 2015

ABSTRACT

Aquaglyceroporins (GlpFs) that transport glycerol along with water and other uncharged solutes are involved in osmoregulation in myriad species. Fungal species form a large group of eukaryotic organisms, and their GlpFs may be diverse, exhibiting various activities. However, few filamentous fungal GlpFs have been biologically investigated. Here, a
glpF
gene from the halophilic fungus
Aspergillus glaucus
(
AgglpF
) was verified to be a channel of water or glycerol in
Xenopus laevis
oocytes and was further functionally analyzed in three heterologous systems. In
Saccharomyces cerevisiae
, cells overexpressing
AgglpF
possessed significant tolerance of drought, salt, and certain metal ions.
AgglpF
was then characterized in the filamentous fungus of
Neurospora crassa
. Based on the
N. crassa
aquaporin gene (
NcAQP
) disruption mutant (the Δ
aqp
mutant), a series of complementary strains carrying
NcAQP
and
AgglpF
and three asparagine-proline-alanine-gene (
NPA
)-deleted
AgglpF
fragments were created. As revealed by salt resistance analysis, the
AgglpF
complementary strain possessed the highest salt resistance among the tested strains. In addition, the intracellular glycerol content in the
AgglpF
complementary strain was markedly higher than that in the other strains. The AgGlpF-green fluorescent protein (GFP) fusion protein was subcellularly localized in the plasma membrane of onion epidermal cells, suggesting that
AgglpF
functions in plants. Indeed, when
AgglpF
was expressed in
Arabidopsis thaliana
, transgenic lines survived under conditions of high osmotic stress and under conditions of drought stress in particular. Overall, our results revealed that AgGlpF as a water/glycerol transporter is required for survival of both fungi and plants under conditions of high osmotic stress and may have value in applications in genetic engineering for generating high salt and drought resistance.
The Octatricopeptide Repeat Protein Raa8 Is Required for Chloroplast
trans
Splicing
Authors: Marx Christina , Wünsch Christiane , Kück Ulrich 
Published: July 2015

ABSTRACT

The mRNA maturation of the tripartite chloroplast
psaA
gene from the green alga
Chlamydomonas reinhardtii
depends on various nucleus-encoded factors that participate in
trans
splicing of two group II introns. Recently, a multiprotein complex was identified that is involved in processing the
psaA
precursor mRNA. Using coupled tandem affinity purification (TAP) and mass spectrometry analyses with the
trans
-splicing factor Raa4 as a bait protein, we recently identified a multisubunit ribonucleoprotein (RNP) complex comprising the previously characterized
trans
-splicing factors Raa1, Raa3, Raa4, and Rat2 plus novel components. Raa1 and Rat2 share a structural motif, an octatricopeptide repeat (OPR), that presumably functions as an RNA interaction module. Two of the novel RNP complex components also exhibit a predicted OPR motif and were therefore considered potential
trans
-splicing factors. In this study, we selected bacterial artificial chromosome (BAC) clones encoding these OPR proteins and conducted functional complementation assays using previously generated
trans
-splicing mutants. Our assay revealed that the
trans
-splicing defect of mutant F19 was restored by a new factor we named
RAA8
; molecular characterization of complemented strains verified that Raa8 participates in splicing of the first
psaA
group II intron. Three of six OPR motifs are located in the C-terminal end of Raa8, which was shown to be essential for restoring
psaA
mRNA
trans
splicing. Our results support the important role played by OPR proteins in chloroplast RNA metabolism and also demonstrate that combining TAP and mass spectrometry with functional complementation studies represents a vigorous tool for identifying
trans
-splicing factors.