bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanical parameters of the molecular motor myosin II determined in permeabilised fibres from slow and fast skeletal muscles of the rabbit
Authors: Percario Valentina , Boncompagni Simona , Protasi Feliciano , Pertici Irene , Pinzauti Francesca , Caremani Marco 
Published: November 2017

Key points
The different performance of slow and fast muscles is mainly attributed to diversity of the myosin heavy chain (MHC) isoform expressed within them.
In this study fast sarcomere‐level mechanics has been applied to Ca2+‐activated single permeabilised fibres isolated from soleus (containing the slow myosin isoform) and psoas (containing the fast myosin isoform) muscles of rabbit for a comparative definition of the mechano‐kinetics of force generation by slow and fast myosin isoforms in situ.
The stiffness and the force of the slow myosin isoform are three times smaller than those of the fast isoform, suggesting that the stiffness of the myosin motor is a determinant of the isoform‐dependent functional diversity between skeletal muscles.
These results open the question of the mechanism that can reconcile the reduced performance of the slow MHC with the higher efficiency of the slow muscle.
AbstractThe skeletal muscle exhibits large functional differences depending on the myosin heavy chain (MHC) isoform expressed in its molecular motor, myosin II. The differences in the mechanical features of force generation by myosin isoforms were investigated in situ by using fast sarcomere‐level mechanical methods in permeabilised fibres (sarcomere length 2.4 μm, temperature 12°C, 4% dextran T‐500) from slow (soleus, containing the MHC‐1 isoform) and fast (psoas, containing the MHC‐2X isoform) skeletal muscle of the rabbit. The stiffness of the half‐sarcomere was determined at the plateau of Ca2+‐activated isometric contractions and in rigor and analysed with a model that accounted for the filament compliance to estimate the stiffness of the myosin motor (ε). ε was 0.56 ± 0.04 and 1.70 ± 0.37 pN nm−1 for the slow and fast isoform, respectively, while the average strain per attached motor (s0) was similar (∼3.3 nm) in both isoforms. Consequently the force per motor (F0 = εs0) was three times smaller in the slow isoform than in the fast isoform (1.89 ± 0.43 versus 5.35 ± 1.51 pN). The fraction of actin‐attached motors responsible for maximum isometric force at saturating Ca2+ (T0,4.5) was 0.47 ± 0.09 in soleus fibres, 70% larger than that in psoas fibres (0.29 ± 0.08), so that F0 in slow fibres was decreased by only 53%. The lower stiffness and force of the slow myosin isoform open the question of the molecular basis of the higher efficiency of slow muscle with respect to fast muscle.
glnA
Truncation in Salmonella enterica Results in a Small Colony Variant Phenotype, Attenuated Host Cell Entry, and Reduced Expression of Flagellin and SPI-1-Associated Effector Genes
Authors: Aurass Philipp , Düvel Juliane , Karste Susanne , Nübel Ulrich , Rabsch Wolfgang , Flieger Antje 
Published: November 2017

ABSTRACT

Many pathogenic bacteria use sophisticated survival strategies to overcome harsh environmental conditions. One strategy is the formation of slow-growing subpopulations termed small colony variants (SCVs). Here we characterize an SCV that spontaneously emerged from an axenic
Salmonella enterica
serovar Typhimurium water culture. We found that the SCV harbored a frameshift mutation in the glutamine synthetase gene
glnA
, leading to an ∼90% truncation of the corresponding protein. Glutamine synthetase, a central enzyme in nitrogen assimilation, converts glutamate and ammonia to glutamine. Glutamine is an important nitrogen donor that is required for the synthesis of cellular compounds. The internal glutamine pool serves as an indicator of nitrogen availability in
Salmonella
. In our study, the SCV and a constructed
glnA
knockout mutant showed reduced growth rates, compared to the wild type. Moreover, the SCV and the
glnA
mutant displayed attenuated entry into host cells and severely reduced levels of exoproteins, including flagellin and several
Salmonella
pathogenicity island 1 (SPI-1)-dependent secreted virulence factors. We found that these proteins were also depleted in cell lysates, indicating their diminished synthesis. Accordingly, the SCV and the
glnA
mutant had severely decreased expression of flagellin genes, several SPI-1 effector genes, and a class 2 motility gene (
flgB
). However, the expression of a class 1 motility gene (
flhD
) was not affected. Supplementation with glutamine or genetic reversion of the
glnA
truncation restored growth, cell entry, gene expression, and protein abundance. In summary, our data show that
glnA
is essential for the growth of
S. enterica
and controls important motility- and virulence-related traits in response to glutamine availability.

IMPORTANCE
Salmonella enterica
serovar Typhimurium is a significant pathogen causing foodborne infections. Here we describe an
S
. Typhimurium small colony variant (SCV) that spontaneously emerged from a long-term starvation experiment in water. It is important to study SCVs because (i) SCVs may arise spontaneously upon exposure to stresses, including environmental and host defense stresses, (ii) SCVs are slow growing and difficult to eradicate, and (iii) only a few descriptions of
S. enterica
SCVs are available. We clarify the genetic basis of the SCV described here as a frameshift mutation in the glutamine synthetase gene
glnA
, leading to glutamine auxotrophy. In
Salmonella
, internal glutamine limitation serves as a sign of external nitrogen deficiency and is thought to regulate cell growth. In addition to exhibiting impaired growth, the SCV showed reduced host cell entry and reduced expression of SPI-1 virulence and flagellin genes.
Temporal Stability of Escherichia coli Concentrations in Waters of Two Irrigation Ponds in Maryland
Authors: Pachepsky Yakov , Kierzewski Rachel , Stocker Matthew , Sellner Kevin , Mulbry Walter , Lee Hoonsoo , Kim Moon 
Published: November 2017

ABSTRACT

Fecal contamination of water sources is an important water quality issue for agricultural irrigation ponds.
Escherichia coli
concentrations are commonly used to evaluate recreational and irrigation water quality. We hypothesized that there may exist temporally stable spatial patterns of
E. coli
concentrations across ponds, meaning that some areas mostly have higher and other areas mostly lower than average concentrations of
E. coli
. To test this hypothesis, we sampled two irrigation ponds in Maryland at nodes of spatial grids biweekly during the summer of 2016. Environmental covariates—temperature, turbidity, conductivity, pH, dissolved oxygen, chlorophyll
a
, and nutrients—were measured in conjunction with
E. coli
concentrations. Temporal stability was assessed using mean relative differences between measurements in each location and averaged measurements across ponds. Temporally stable spatial patterns of
E. coli
concentrations and the majority of environmental covariates were expressed for both ponds. In the pond interior, larger relative mean differences in chlorophyll
a
corresponded to smaller mean relative differences in
E. coli
concentrations, with a Spearman's rank correlation coefficient of 0.819. Turbidity and ammonium concentrations were the two other environmental covariates with the largest positive correlations between their location ranks and the
E. coli
concentration location ranks. Tenfold differences were found between geometric mean
E. coli
concentrations in locations that were consistently high or consistently low. The existence of temporally stable patterns of
E. coli
concentrations can affect the results of microbial water quality assessment in ponds and should be accounted for in microbial water quality monitoring design.

IMPORTANCE
The microbial quality of water in irrigation water sources must be assessed to prevent the spread of microbes that can cause disease in humans because of produce consumption. The microbial quality of irrigation water is evaluated based on concentrations of
Escherichia coli
as the indicator organism. Given the high spatial and temporal variability of
E. coli
concentrations in irrigation water sources, recommendations are needed on where and when samples of water have to be taken for microbial analysis. This work demonstrates the presence of a temporally stable spatial pattern in the distributions of
E. coli
concentrations across irrigation ponds. The ponds studied had zones where
E. coli
concentrations were mostly higher than average and zones where the concentrations were mostly lower than average over the entire observation period, covering the season when water was used for irrigation. Accounting for the existence of such zones will improve the design and implementation of microbial water quality monitoring.
Differential RNA Sequencing Implicates Sulfide as the Master Regulator of S
0
Metabolism in Chlorobaculum tepidum and Other Green Sulfur Bacteria
Authors: Hilzinger Jacob M. , Raman Vidhyavathi , Shuman Kevin E. , Eddie Brian J. , Hanson Thomas E. 
Published: November 2017

ABSTRACT

The green sulfur bacteria (
Chlorobiaceae
) are anaerobes that use electrons from reduced sulfur compounds (sulfide, S
0
, and thiosulfate) as electron donors for photoautotrophic growth.
Chlorobaculum tepidum
, the model system for the
Chlorobiaceae
, both produces and consumes extracellular S
0
globules depending on the availability of sulfide in the environment. These physiological changes imply significant changes in gene regulation, which has been observed when sulfide is added to
Cba. tepidum
growing on thiosulfate. However, the underlying mechanisms driving these gene expression changes, i.e., the specific regulators and promoter elements involved, have not yet been defined. Here, differential RNA sequencing (dRNA-seq) was used to globally identify transcript start sites (TSS) that were present during growth on sulfide, biogenic S
0
, and thiosulfate as sole electron donors. TSS positions were used in combination with RNA-seq data from cultures growing on these same electron donors to identify both basal promoter elements and motifs associated with electron donor-dependent transcriptional regulation. These motifs were conserved across homologous
Chlorobiaceae
promoters. Two lines of evidence suggest that sulfide-mediated repression is the dominant regulatory mode in
Cba. tepidum
. First, motifs associated with genes regulated by sulfide overlap key basal promoter elements. Second, deletion of the
Cba. tepidum
1277
(
CT1277
) gene, encoding a putative regulatory protein, leads to constitutive overexpression of the sulfide:quinone oxidoreductase CT1087 in the absence of sulfide. The results suggest that sulfide is the master regulator of sulfur metabolism in
Cba. tepidum
and the
Chlorobiaceae
. Finally, the identification of basal promoter elements with differing strengths will further the development of synthetic biology in
Cba. tepidum
and perhaps other
Chlorobiaceae
.

IMPORTANCE
Elemental sulfur is a key intermediate in biogeochemical sulfur cycling. The photoautotrophic green sulfur bacterium
Chlorobaculum tepidum
either produces or consumes elemental sulfur depending on the availability of sulfide in the environment. Our results reveal transcriptional dynamics of
Chlorobaculum tepidum
on elemental sulfur and increase our understanding of the mechanisms of transcriptional regulation governing growth on different reduced sulfur compounds. This report identifies genes and sequence motifs that likely play significant roles in the production and consumption of elemental sulfur. Beyond this focused impact, this report paves the way for the development of synthetic biology in
Chlorobaculum tepidum
and other
Chlorobiaceae
by providing a comprehensive identification of promoter elements for control of gene expression, a key element of strain engineering.
Cry64Ba and Cry64Ca, Two ETX/MTX2-Type Bacillus thuringiensis Insecticidal Proteins Active against Hemipteran Pests
Authors: Liu Yonglei , Wang Yinglong , Shu Changlong , Lin Kejian , Song Fuping , Bravo Alejandra , Soberón Mario , Zhang Jie 
Published: November 2017

ABSTRACT

Genetically modified crops that express insecticidal
Bacillus thuringiensis
(Bt) proteins have become a primary approach for control of lepidopteran (moth) and coleopteran (beetle) pests that feed by chewing the plants. However, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. In this study, we describe two Cry toxins (Cry64Ba and Cry64Ca) from Bt strain 1012 that showed toxicity against two important hemipteran rice pests,
Laodelphax striatellus
and
Sogatella furcifera
. Both of these proteins contain an ETX/MTX2 domain and share common sequence features with the β-pore-forming toxins. Coexpression of
cry64Ba
and
cry64Ca
genes in the acrystalliferous Bt strain HD73
−
resulted in high insecticidal activity against both hemipteran pests. No toxicity was observed on other pests such as
Ostrinia furnacalis
,
Plutella xylostella
, or
Colaphellus bowringi
. Also, no hemolytic activity or toxicity against cancer cells was detected. Binding assays showed specific binding of the Cry64Ba/Cry64Ca toxin complex to brush border membrane vesicles isolated from
L. striatellus
. Cry64Ba and Cry64Ca are Bt Cry toxins highly effective against hemipteran pests and could provide a novel strategy for the environmentally friendly biological control of rice planthoppers in transgenic plants.

IMPORTANCE
In Asia, rice is an important staple food, whose production is threatened by rice planthoppers. To date, no effective
Bacillus thuringiensis
(Bt) protein has been shown to have activity against rice planthoppers. We cloned two Bt toxin genes from Bt strain 1012 that showed toxicity against small brown planthoppers (
Laodelphax striatellus
) and white-backed planthoppers (
Sogatella furcifera
). To our knowledge, the proteins encoded by the
cry64Ba
and
cry64Ca
genes are the most efficient insecticidal Bt Cry proteins with activity against hemipteran insects reported so far. Cry64Ba and Cry64Ca showed no toxicity against some lepidopteran or coleopteran pests. These two proteins should be able to be used for integrated hemipteran pest management.
Diversity of Three-Dimensional Structures and Catalytic Mechanisms of Alginate Lyases
Authors: Xu Fei , Wang Peng , Zhang Yu-Zhong , Chen Xiu-Lan 
Published: November 2017

ABSTRACT

Alginate is a linear polysaccharide produced mainly by brown algae in marine environments. Alginate consists of a linear block copolymer made up of two monomeric units, β-
d
-mannuronate (M) and its C-5 epimer α-
l
-guluronate (G). Alginate lyases are polysaccharide lyases (PL) that degrade alginate via a β-elimination reaction. These enzymes play an important role in marine carbon recycling and also have widespread industrial applications. So far, more than 1,774 alginate lyase sequences have been identified and are distributed into 7 PL families. In this review, the folds, conformational changes during catalysis, and catalytic mechanisms of alginate lyases are described. Thus far, structures for 15 alginate lyases have been solved and are divided into 3 fold classes: the β-jelly roll class (PL7, -14, and -18), the (α/α)n toroid class (PL5, -15, and -17), and the β-helix fold (PL6). These enzymes adopt two different mechanisms for catalysis, and three kinds of conformational changes occur during this process. Moreover, common features in the structures, conformational changes, and catalytic mechanisms are summarized, providing a comprehensive understanding on alginate lyases.
High Prevalence and Genetic Diversity of Large phiCD211 (phiCDIF1296T)-Like Prophages in Clostridioides difficile
Authors: Garneau Julian R. , Sekulovic Ognjen , Dupuy Bruno , Soutourina Olga , Monot Marc , Fortier Louis-Charles 
Published: November 2017

ABSTRACT

Clostridioides difficile
(formerly
Clostridium difficile
) is a pathogenic bacterium displaying great genetic diversity. A significant proportion of this diversity is due to the presence of integrated prophages. Here, we provide an in-depth analysis of phiCD211, also known as phiCDIF1296T, the largest phage identified in
C. difficile
so far, with a genome of 131 kbp. It shares morphological and genomic similarity with other large siphophages, like phage 949, infecting
Lactococcus lactis
, and phage c-st, infecting
Clostridium botulinum
. A PhageTerm analysis indicated the presence of 378-bp direct terminal repeats at the phiCD211 genome termini. Among striking features of phiCD211, the presence of several transposase and integrase genes suggests past recombination events with other mobile genetic elements. Several gene products potentially influence the bacterial lifestyle and fitness, including a putative AcrB/AcrD/AcrF multidrug resistance protein, an EzrA septation ring formation regulator, and a spore protease. We also identified a CRISPR locus and a
cas3
gene. We screened 2,584
C. difficile
genomes available and detected 149 prophages sharing ≥80% nucleotide identity with phiCD211 (5% prevalence). Overall, phiCD211-like phages were detected in
C. difficile
strains corresponding to 21 different multilocus sequence type groups, showing their high prevalence. Comparative genomic analyses revealed the existence of several clusters of highly similar phiCD211-like phages. Of note, large chromosome inversions were observed in some members, as well as multiple gene insertions and module exchanges. This highlights the great plasticity and gene coding potential of the phiCD211/phiCDIF1296T genome. Our analyses also suggest active evolution involving recombination with other mobile genetic elements.

IMPORTANCE
Clostridioides difficile
is a clinically important pathogen representing a serious threat to human health. Our hypothesis is that genetic differences between strains caused by the presence of integrated prophages could explain the apparent differences observed in the virulence of different
C. difficile
strains. In this study, we provide a full characterization of phiCD211, also known as phiCDIF1296T, the largest phage known to infect
C. difficile
so far. Screening 2,584
C. difficile
genomes revealed the presence of highly similar phiCD211-like phages in 5% of the strains analyzed, showing their high prevalence. Multiple-genome comparisons suggest that evolution of the phiCD211-like phage community is dynamic, and some members have acquired genes that could influence bacterial biology and fitness. Our study further supports the relevance of studying phages in
C. difficile
to better understand the epidemiology of this clinically important human pathogen.
Loop of Streptomyces Feruloyl Esterase Plays an Important Role in the Enzyme's Catalyzing the Release of Ferulic Acid from Biomass
Authors: Uraji Misugi , Tamura Haruka , Mizohata Eiichi , Arima Jiro , Wan Kun , Ogawa Ken'ichi , Inoue Tsuyoshi , Hatanaka Tadashi 
Published: November 2017

ABSTRACT

Feruloyl esterases (FAEs) are key enzymes required for the production of ferulic acid from agricultural biomass. Previously, we identified and characterized R18, an FAE from
Streptomyces cinnamoneus
NBRC 12852, which showed no sequence similarity to the known FAEs. To determine the region involved in its catalytic activity, we constructed chimeric enzymes using R18 and its homolog (TH2-18) from
S. cinnamoneus
strain TH-2. Although R18 and TH2-18 showed 74% identity in their primary sequences, the recombinant proteins of these two FAEs (recombinant R18 [rR18] and rTH2-18) showed very different specific activities toward ethyl ferulate. By comparing the catalytic activities of the chimeras, a domain comprised of residues 140 to 154 was found to be crucial for the catalytic activity of R18. Furthermore, we analyzed the crystal structure of rR18 at a resolution of 1.5 Å to elucidate the relationship between its activity and its structure. rR18 possessed a typical catalytic triad, consisting of Ser-191, Asp-214, and His-268, which was characteristic of the serine esterase family. By structural analysis, the above-described domain was found to be present in a loop-like structure (the R18 loop), which possessed a disulfide bond conserved in the genus
Streptomyces
. Moreover, compared to rTH2-18 of its parental strain, the TH2-18 mutant, in which Pro and Gly residues were inserted into the domain responsible for forming the R18 loop, showed markedly high
k
cat
values using artificial substrates. We also showed that the FAE activity of TH2-18 toward corn bran, a natural substrate, was improved by the insertion of the Gly and Pro residues.

IMPORTANCE
Streptomyces
species are widely distributed bacteria that are predominantly present in soil and function as decomposers in natural environments. They produce various enzymes, such as carbohydrate hydrolases, esterases, and peptidases, which decompose agricultural biomass. In this study, based on the genetic information on two
Streptomyces cinnamoneus
strains, we identified novel feruloyl esterases (FAEs) capable of producing ferulic acid from biomass. These two FAEs shared high similarity in their amino acid sequences but did not resemblance any known FAEs. By comparing chimeric proteins and performing crystal structure analysis, we confirmed that a flexible loop was important for the catalytic activity of
Streptomyces
FAEs. Furthermore, we determined that the catalytic activity of one FAE was improved drastically by inserting only 2 amino acids into its loop-forming domain. Thus, differences in the amino acid sequence of the loop resulted in different catalytic activities. In conclusion, our findings provide a foundation for the development of novel enzymes for industrial use.
Draft Whole-Genome Sequences of 18
Flavobacterium
spp
Authors: Stine Cynthia B. , Li Cong , Crosby Tina C. , Hasbrouck Nicholas R. , Lam Claudia , Tadesse Daniel A. 
Published: November 2017

ABSTRACT

We report here the draft whole-genome sequences for 18
Flavobacterium
species type strains that have historically been associated with fish gill disease.
Genome Sequences of 19 Novel Erwinia amylovora Bacteriophages
Authors: Esplin Ian N. D. , Berg Jordan A. , Sharma Ruchira , Allen Robert C. , Arens Daniel K. , Ashcroft Cody R. , Bairett Shannon R. , Beatty Nolan J. , Bickmore Madeline , Bloomfield Travis J. , Brady T. Scott , Bybee Rachel N. , Carter John L. , Choi Minsey C. , Duncan Steven , Fajardo Christopher P. , Foy Brayden B. , Fuhriman David A. , Gibby Paul D. , Grossarth Savannah E. , Harbaugh Kala , Harris Natalie , Hilton Jared A. , Hurst Emily , Hyde Jonathan R. , Ingersoll Kayleigh , Jacobson Caitlin M. , James Brady D. , Jarvis Todd M. , Jaen-Anieves Daniella , Jensen Garrett L. , Knabe Bradley K. , Kruger Jared L. , Merrill Bryan D. , Pape Jenny A. , Payne Anderson Ashley M. , Payne David E. , Peck Malia D. , Pollock Samuel V. , Putnam Micah J. , Ransom Ethan K. , Ririe Devin B. , Robinson David M. , Rogers Spencer L. , Russell Kerri A. , Schoenhals Jonathan E. , Shurtleff Christopher A. , Simister Austin R. , Smith Hunter G. , Stephenson Michael B. , Staley Lyndsay A. , Stettler Jason M. , Stratton Mallorie L. , Tateoka Olivia B. , Tatlow P. J. , Taylor Alexander S. , Thompson Suzanne E. , Townsend Michelle H. , Thurgood Trever L. , Usher Brittian K. , Whitley Kiara V. , Ward Andrew T. , Ward Megan E. H. , Webb Charles J. , Wienclaw Trevor M. , Williamson Taryn L. , Wells Michael J. , Wright Cole K. , Breakwell Donald P. , Hope Sandra , Grose Julianne H. 
Published: November 2017

ABSTRACT

Erwinia amylovora
is the causal agent of fire blight, a devastating disease affecting some plants of the
Rosaceae
family. We isolated bacteriophages from samples collected from infected apple and pear trees along the Wasatch Front in Utah. We announce 19 high-quality complete genome sequences of
E. amylovora
bacteriophages.