bluuebunny/crossref_metadata_2025_split huggingface dataset:
Gene Deletion Strategy To Examine the Involvement of the Two Chondroitin Lyases in Flavobacterium columnare Virulence
Authors: Li Nan , Qin Ting , Zhang Xiao Lin , Huang Bei , Liu Zhi Xin , Xie Hai Xia , Zhang Jin , McBride Mark J. , Nie Pin 
Published: August 2015

ABSTRACT

Flavobacterium columnare
is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The
F. johnsoniae
ompA
promoter (P
ompA
) was fused to
sacB
to construct a counterselectable marker for
F. columnare
.
F. columnare
carrying P
ompA-sacB
failed to grow on media containing 10% sucrose. A suicide vector carrying P
ompA-sacB
was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes,
cslA
and
cslB
, were deleted. The Δ
cslA
and Δ
cslB
mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (Δ
cslA
Δ
cslB
) was completely deficient in chondroitin lyase activity. Cells of
F. columnare
wild-type strain G
4
and of the chondroitin lyase-deficient Δ
cslA
Δ
cslB
mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of
F. columnare
but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum
Bacteroidetes
.
Comparative Analysis of Extremely Thermophilic Caldicellulosiruptor Species Reveals Common and Unique Cellular Strategies for Plant Biomass Utilization
Authors: Zurawski Jeffrey V. , Conway Jonathan M. , Lee Laura L. , Simpson Hunter J. , Izquierdo Javier A. , Blumer-Schuette Sara , Nookaew Intawat , Adams Michael W. W. , Kelly Robert M. 
Published: August 2015

ABSTRACT

Microbiological, genomic and transcriptomic analyses were used to examine three species from the bacterial genus
Caldicellulosiruptor
with respect to their capacity to convert the carbohydrate content of lignocellulosic biomass at 70°C to simple sugars, acetate, lactate, CO
2
, and H
2
.
Caldicellulosiruptor bescii
,
C. kronotskyensis
, and
C. saccharolyticus
solubilized 38%, 36%, and 29% (by weight) of unpretreated switchgrass (
Panicum virgatum
) (5 g/liter), respectively, which was about half of the amount of crystalline cellulose (Avicel; 5 g/liter) that was solubilized under the same conditions. The lower yields with
C. saccharolyticus
, not appreciably greater than the thermal control for switchgrass, were unexpected, given that its genome encodes the same glycoside hydrolase 9 (GH9)-GH48 multidomain cellulase (CelA) found in the other two species. However, the genome of
C. saccharolyticus
lacks two other cellulases with GH48 domains, which could be responsible for its lower levels of solubilization. Transcriptomes for growth of each species comparing cellulose to switchgrass showed that many carbohydrate ABC transporters and multidomain extracellular glycoside hydrolases were differentially regulated, reflecting the heterogeneity of lignocellulose. However, significant differences in transcription levels for conserved genes among the three species were noted, indicating unexpectedly diverse regulatory strategies for deconstruction for these closely related bacteria. Genes encoding the Che-type chemotaxis system and flagellum biosynthesis were upregulated in
C. kronotskyensis
and
C. bescii
during growth on cellulose, implicating motility in substrate utilization. The results here show that capacity for plant biomass deconstruction varies across
Caldicellulosiruptor
species and depends in a complex way on GH genome inventory, substrate composition, and gene regulation.
Identification of the Gene Cluster for the Anaerobic Degradation of 3,5-Dihydroxybenzoate (α-Resorcylate) in Thauera aromatica Strain AR-1
Authors: Molina-Fuentes Águeda , Pacheco Daniel , Marín Patricia , Philipp Bodo , Schink Bernhard , Marqués Silvia 
Published: August 2015

ABSTRACT

Thauera aromatica
strain AR-1 degrades 3,5-dihydroxybenzoate (3,5-DHB) with nitrate as an electron acceptor. Previous biochemical studies have shown that this strain converts 3,5-DHB to hydroxyhydroquinone (1,2,4-trihydroxybenzene) through water-dependent hydroxylation of the aromatic ring and subsequent decarboxylation, and they suggest a pathway homologous to that described for the anaerobic degradation of 1,3-dihydroxybenzene (resorcinol) by
Azoarcus anaerobius
. Southern hybridization of a
T. aromatica
strain AR-1 gene library identified a 25-kb chromosome region based on its homology with
A. anaerobius
main pathway genes. Sequence analysis defined 20 open reading frames. Knockout mutations of the most relevant genes in the pathway were generated by reverse genetics. Physiological and biochemical analyses identified the genes for the three main steps in the pathway which were homologous to those described in
A. anaerobius
and suggested the function of several auxiliary genes possibly involved in enzyme maturation and intermediate stabilization. However,
T. aromatica
strain AR-1 had an additional enzyme to metabolize hydroxyhydroquinone, a putative cytoplasmic quinone oxidoreductase. In addition, a specific tripartite ATP-independent periplasmic (TRAP) transport system was required for efficient growth on 3,5-DHB. Reverse transcription-PCR (RT-PCR) analysis showed that the pathway genes were organized in five 3,5-DHB-inducible operons, three of which have been shown to be under the control of a single LysR-type transcriptional regulator, DbdR. Despite sequence homology, the genetic organizations of the clusters in
T. aromatica
strain AR-1 and
A. anaerobius
differed substantially.
The Hybrid Pre-CTXΦ-RS1 Prophage Genome and Its Regulatory Function in Environmental Vibrio cholerae O1 Strains
Authors: Wang Hongxia , Pang Bo , Xiong Lifeng , Wang Duochun , Wang Xiaomei , Zhang Lijuan , Kan Biao 
Published: August 2015

ABSTRACT

The cholera toxin genes of
Vibrio cholerae
are encoded by CTXΦ, a lysogenic bacteriophage. Infection with this phage plays a determinant role in toxigenicity conversion and the emergence of new clones of pathogenic
V. cholerae
. Multiple phage alleles, defined by sequence types of the repressor gene
rstR
, have been found, showing the divergence of phage genomes. Pre-CTXΦ, which is characterized by the absence of toxin genes, is predicted to be the precursor of CTXΦ. We have found a new pre-CTXΦ prophage genome (named pre-CTX
ZJ
Φ for its novel
rstR
allele) in nontoxigenic
V. cholerae
O1 isolates that were obtained during surveillance of the estuary water of the Zhujiang River. A novel hybrid genome of the helper phage RS1 was identified in an environmental strain carrying pre-CTX
ZJ
Φ in this study. The chromosomal integration and genomic arrangement of pre-CTX
ZJ
Φ and RS1 were determined. The RS2 of pre-CTX
ZJ
Φ was shown to have a function in replication, but it seemed to have lost its ability to integrate. The RstR of pre-CTX
ZJ
Φ exerted the highest repression of its own
rstA
promoter compared to other RstRs, suggesting
rstR
-specific phage superinfection immunity and potential coinfection with other pre-CTXΦ/CTXΦ alleles. The environmental strain carrying pre-CTX
ZJ
Φ could still be infected by CTX
ET
Φ, the most common phage allele in the strains of the seventh cholera pandemic, suggesting that this nontoxigenic clone could potentially undergo toxigenicity conversion by CTXΦ infection and become a new toxigenic clone despite already containing the pre-CTXΦ prophage.
Biochemical Characterization of the Lactobacillus reuteri Glycoside Hydrolase Family 70 GTFB Type of 4,6-α-Glucanotransferase Enzymes That Synthesize Soluble Dietary Starch Fibers
Authors: Bai Yuxiang , van der Kaaij Rachel Maria , Leemhuis Hans , Pijning Tjaard , van Leeuwen Sander Sebastiaan , Jin Zhengyu , Dijkhuizen Lubbert 
Published: August 2015

ABSTRACT

4,6-α-Glucanotransferase (4,6-α-GTase) enzymes, such as GTFB and GTFW of
Lactobacillus reuteri
strains, constitute a new reaction specificity in glycoside hydrolase family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/maltopolysaccharides (IMMPs). These IMMPs still have linear chains with some α1→4 linkages but mostly (relatively long) linear chains with α1→6 linkages and are soluble dietary starch fibers. 4,6-α-GTase enzymes and their products have significant potential for industrial applications. Here we report that an N-terminal truncation (amino acids 1 to 733) strongly enhances the soluble expression level of fully active GTFB-ΔN (approximately 75-fold compared to full-length wild type GTFB) in
Escherichia coli
. In addition, quantitative assays based on amylose V as the substrate are described; these assays allow accurate determination of both hydrolysis (minor) activity (glucose release, reducing power) and total activity (iodine staining) and calculation of the transferase (major) activity of these 4,6-α-GTase enzymes. The data show that GTFB-ΔN is clearly less hydrolytic than GTFW, which is also supported by nuclear magnetic resonance (NMR) analysis of their final products. From these assays, the biochemical properties of GTFB-ΔN were characterized in detail, including determination of kinetic parameters and acceptor substrate specificity. The GTFB enzyme displayed high conversion yields at relatively high substrate concentrations, a promising feature for industrial application.
Dynamic Mechanisms of the Bactericidal Action of an Al
2
O
3
-TiO
2
-Ag Granular Material on an Escherichia coli Strain
Authors: Tartanson Marie-Anne , Soussan Laurence , Rivallin Matthieu , Pecastaings Sophie , Chis Cristian V. , Penaranda Diego , Roques Christine , Faur Catherine 
Published: August 2015

ABSTRACT

The bactericidal activity of an Al
2
O
3
-TiO
2
-Ag granular material against an
Escherichia coli
strain was confirmed by a culture-based method. In particular, 100% of microorganisms were permanently inactivated in 30 to 45 min. The present work aimed to investigate the mechanisms of the bactericidal action of this material and their dynamics on
Escherichia coli
using different techniques. Observations by transmission electron microscopy (TEM) at different times of disinfection revealed morphological changes in the bacteria as soon as they were put in contact with the material. Notably highlighted were cell membrane damage; cytoplasm detachment; formation of vacuoles, possibly due to DNA condensation, in association with regions exhibiting different levels of electron density; and membrane lysis. PCR and flow cytometry analyses were used to confirm and quantify the observations of cell integrity. The direct exposure of cells to silver, combined with the oxidative stress induced by the reactive oxygen species (ROS) generated, was identified to be responsible for these morphological alterations. From the first 5 min of treatment with the Al
2
O
3
-TiO
2
-Ag material, 98% of
E. coli
isolates were lysed. From 30 min, cell viability decreased to reach total inactivation, although approximately 1% of permeable
E. coli
cells and 1% of intact cells (10
5
genomic units · ml
−1
) were evidenced. This study demonstrates that the bactericidal effect of the material results from a synergic action of desorbed and supported silver. Supported silver was shown to generate the ROS evidenced.
Scarless Genome Editing and Stable Inducible Expression Vectors for Geobacter sulfurreducens
Authors: Chan Chi Ho , Levar Caleb E. , Zacharoff Lori , Badalamenti Jonathan P. , Bond Daniel R. 
Published: August 2015

ABSTRACT

Metal reduction by members of the
Geobacteraceae
is encoded by multiple gene clusters, and the study of extracellular electron transfer often requires biofilm development on surfaces. Genetic tools that utilize polar antibiotic cassette insertions limit mutant construction and complementation. In addition, unstable plasmids create metabolic burdens that slow growth, and the presence of antibiotics such as kanamycin can interfere with the rate and extent of
Geobacter
biofilm growth. We report here genetic system improvements for the model anaerobic metal-reducing bacterium
Geobacter sulfurreducens
. A motile strain of
G. sulfurreducens
was constructed by precise removal of a transposon interrupting the
fgrM
flagellar regulator gene using SacB/sucrose counterselection, and Fe(III) citrate reduction was eliminated by deletion of the gene encoding the inner membrane cytochrome
imcH
. We also show that RK2-based plasmids were maintained in
G. sulfurreducens
for over 15 generations in the absence of antibiotic selection in contrast to unstable pBBR1 plasmids. Therefore, we engineered a series of new RK2 vectors containing native constitutive
Geobacter
promoters, and modified one of these promoters for VanR-dependent induction by the small aromatic carboxylic acid vanillate. Inducible plasmids fully complemented Δ
imcH
mutants for Fe(III) reduction, Mn(IV) oxide reduction, and growth on poised electrodes. A real-time, high-throughput Fe(III) citrate reduction assay is described that can screen numerous
G. sulfurreducens
strain constructs simultaneously and shows the sensitivity of
imcH
expression by the vanillate system. These tools will enable more sophisticated genetic studies in
G. sulfurreducens
without polar insertion effects or need for multiple antibiotics.
Effects of Formulation on Microbicide Potency and Mitigation of the Development of Bacterial Insusceptibility
Authors: Cowley Nicola L. , Forbes Sarah , Amézquita Alejandro , McClure Peter , Humphreys Gavin J. , McBain Andrew J. 
Published: August 2015

ABSTRACT
Risk assessments of the potential for microbicides to select for reduced bacterial susceptibility have been based largely on data generated through the exposure of bacteria to microbicides in aqueous solution. Since microbicides are normally formulated with multiple excipients, we have investigated the effect of formulation on antimicrobial activity and the induction of bacterial insusceptibility. We tested 8 species of bacteria (7 genera) before and after repeated exposure (14 passages), using a previously validated gradient plating system, for their susceptibilities to the microbicides benzalkonium chloride, benzisothiozolinone, chlorhexidine, didecyldimethyl ammonium chloride, DMDM-hydantoin, polyhexamethylene biguanide, thymol, and triclosan in aqueous solution (nonformulated) and in formulation with excipients often deployed in consumer products. Susceptibilities were also assessed following an additional 14 passages without microbicide to determine the stability of any susceptibility changes. MICs and minimum bactericidal concentrations (MBC) were on average 11-fold lower for formulated microbicides than for nonformulated microbicides. After exposure to the antimicrobial compounds, of 72 combinations of microbicide and bacterium there were 19 ≥4-fold (mean, 8-fold) increases in MIC for nonformulated and 8 ≥4-fold (mean, 2-fold) increases in MIC for formulated microbicides. Furthermore, there were 20 ≥4-fold increases in MBC (mean, 8-fold) for nonformulated and 10 ≥4-fold (mean, 2-fold) increases in MBC for formulated microbicides. Susceptibility decreases fully or partially reverted back to preexposure values for 49% of MICs and 72% of MBCs after further passage. In summary, formulated microbicides exhibited greater antibacterial potency than unformulated actives and susceptibility decreases after repeated exposure were lower in frequency and extent.
Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method
Authors: Beilharz Katrin , van Raaphorst Renske , Kjos Morten , Veening Jan-Willem 
Published: August 2015

ABSTRACT

During the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogen
Streptococcus pneumoniae
and other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) in
S. pneumoniae
. Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP in
S. pneumoniae
and is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling in
S. pneumoniae
and related organisms.
A Model of Extracellular Enzymes in Free-Living Microbes: Which Strategy Pays Off?
Authors: Traving Sachia J. , Thygesen Uffe H. , Riemann Lasse , Stedmon Colin A. 
Published: August 2015

ABSTRACT
An initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy entails potential substrates being reduced to very low concentrations. Free enzymes, on the other hand, generate a radically different substrate field, which suggests significant benefits for the strategy if free cells engage in social foraging or experience high substrate concentrations. Swimming has a slight positive effect for the attached-enzyme strategy, while the effect is negative for the free-enzyme strategy. The results of this study suggest that specific dissolved organic compounds in the ocean likely persist below a threshold concentration impervious to biological utilization. This could help explain the persistence and apparent refractory state of oceanic dissolved organic matter (DOM). Microbial extracellular enzyme strategies, therefore, have important implications for larger-scale processes, such as shaping the role of DOM in ocean carbon sequestration.
Semirational Directed Evolution of Loop Regions in Aspergillus japonicus β-Fructofuranosidase for Improved Fructooligosaccharide Production
Authors: Trollope K. M. , Görgens J. F. , Volschenk H. 
Published: August 2015

ABSTRACT

The
Aspergillus japonicus
β-fructofuranosidase catalyzes the industrially important biotransformation of sucrose to fructooligosaccharides. Operating at high substrate loading and temperatures between 50 and 60°C, the enzyme activity is negatively influenced by glucose product inhibition and thermal instability. To address these limitations, the solvent-exposed loop regions of the β-fructofuranosidase were engineered using a combined crystal structure- and evolutionary-guided approach. This semirational approach yielded a functionally enriched first-round library of 36 single-amino-acid-substitution variants with 58% retaining activity, and of these, 71% displayed improved activities compared to the parent. The substitutions yielding the five most improved variants subsequently were exhaustively combined and evaluated. A four-substitution combination variant was identified as the most improved and reduced the time to completion of an efficient industrial-like reaction by 22%. Characterization of the top five combination variants by isothermal denaturation assays indicated that these variants displayed improved thermostability, with the most thermostable variant displaying a 5.7°C increased melting temperature. The variants displayed uniquely altered, concentration-dependent substrate and product binding as determined by differential scanning fluorimetry. The altered catalytic activity was evidenced by increased specific activities of all five variants, with the most improved variant doubling that of the parent. Variant homology modeling and computational analyses were used to rationalize the effects of amino acid changes lacking direct interaction with substrates. Data indicated that targeting substitutions to loop regions resulted in improved enzyme thermostability, specific activity, and relief from product inhibition.
Impacts of Long-Term Irrigation of Domestic Treated Wastewater on Soil Biogeochemistry and Bacterial Community Structure
Authors: Wafula Denis , White John R. , Canion Andy , Jagoe Charles , Pathak Ashish , Chauhan Ashvini 
Published: August 2015

ABSTRACTFreshwater scarcity and regulations on wastewater disposal have necessitated the reuse of treated wastewater (TWW) for soil irrigation, which has several environmental and economic benefits. However, TWW irrigation can cause nutrient loading to the receiving environments. We assessed bacterial community structure and associated biogeochemical changes in soil plots irrigated with nitrate-rich TWW (referred to as pivots) for periods ranging from 13 to 30 years. Soil cores (0 to 40 cm) were collected in summer and winter from five irrigated pivots and three adjacently located nonirrigated plots. Total bacterial and denitrifier gene abundances were estimated by quantitative PCR (qPCR), and community structure was assessed by 454 massively parallel tag sequencing (MPTS) of small-subunit (SSU) rRNA genes along with terminal restriction fragment length polymorphism (T-RFLP) analysis ofnirK,nirS, andnosZfunctional genes responsible for denitrification of the TWW-associated nitrate. Soil physicochemical analyses showed that, regardless of the seasons, pH and moisture contents (MC) were higher in the irrigated (IR) pivots than in the nonirrigated (NIR) plots; organic matter (OM) and microbial biomass carbon (MBC) were higher as a function of season but not of irrigation treatment. MPTS analysis showed that TWW loading resulted in the following: (i) an increase in the relative abundance ofProteobacteria, especiallyBetaproteobacteriaandGammaproteobacteria; (ii) a decrease in the relative abundance ofActinobacteria; (iii) shifts in the communities of acidobacterial groups, along with a shift in thenirKandnirSdenitrifier guilds as shown by T-RFLP analysis. Additionally, bacterial biomass estimated by genus/group-specific real-time qPCR analyses revealed that higher numbers of total bacteria,Acidobacteria,Actinobacteria,Alphaproteobacteria, and thenirSdenitrifier guilds were present in the IR pivots than in the NIR plots. Identification of thenirK-containing microbiota as a proxy for the denitrifier community indicated that bacteria belonged to alphaproteobacteria from theRhizobiaceaefamily within the agroecosystem studied. Multivariate statistical analyses further confirmed some of the above soil physicochemical and bacterial community structure changes as a function of long-term TWW application within this agroecosystem.