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Structures, compositions and activities of live Shewanella biofilms formed on graphite electrodes in electrochemical flow cells.
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Structures, compositions and activities of live Shewanella biofilms formed on graphite electrodes in electrochemical flow cells.

Appl Environ Microbiol. 2017 Jun 16;:

Authors: Kitayama M, Koga R, Kasai T, Kouzuma A, Watanabe K

Abstract
An electrochemical flow cell equipped with a graphite working electrode (WE) at the bottom was inoculated with Shewanella oneidensis MR-1 expressing an anaerobic fluorescence protein, and biofilm formation on WE were observed over time during current generation at WE potentials of +0.4 and 0 V (vs. standard hydrogen electrodes) under electrolyte-flow conditions. Electrochemical analyses suggest the presence of unique electron-transfer mechanisms in the +0.4 V biofilm. Microscopic analyses revealed that, different from aerobic biofilms, current-generating biofilm (at +0.4 V) was thin and flat (∼10 μm in thickness), and cells were evenly and densely distributed in the biofilm. In contrast, cells were unevenly distributed in biofilm formed at 0 V. In situ fluorescent-staining and biofilm-recovery experiments showed that extracellular polysaccharides (EPSs) in the +0.4 V biofilm were much less than those in the aerobic and 0 V biofilms, suggesting that Shewanella cells suppress the production of EPSs at +0.4 V under flow conditions. We suggest that Shewanella cells perceive electrode potentials and modulate the structure and composition of biofilms for efficiently transferring electrons to electrodes.IMPORTANCE A promising application of microbial fuel cells (MFCs) is to save energy for wastewater treatment. Since current is generated in these MFCs by biofilm microbes under horizontal flows of wastewater, it is important to understand mechanisms for biofilm formation and current generation under water-flow conditions. Although massive work has been done to analyze molecular mechanisms for current generation by model exoelectrogenic bacteria, such as Shewanella oneidensis, limited information is available for the formation of current-generating biofilms over time under water-flow conditions. The present study developed electrochemical flow cells and used them to examine electrochemical and structural features of current-generating biofilms under water-flow conditions. We show unique features of mature biofilms actively generating current, which create opportunities to search for as-yet-undiscovered current-generating mechanisms in Shewanella biofilms. Furthermore, information provided in the present study is useful for researchers who attempt to develop anode architectures suitable for wastewater-treatment MFCs.

PMID: 28625998 [PubMed - as supplied by publisher]




Quantifying Vibrio cholerae enterotoxicity in a zebrafish infection model.
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Quantifying Vibrio cholerae enterotoxicity in a zebrafish infection model.

Appl Environ Microbiol. 2017 Jun 16;:

Authors: Mitchell KC, Breen P, Britton S, Neely MN, Withey JH

Abstract
Vibrio cholerae is the etiological agent of cholera, an acute intestinal infection in humans characterized by voluminous watery diarrhea. Cholera is spread through ingestion of contaminated food or water, primarily in developing countries that lack the proper infrastructure for proper water and sewage treatment. Vibrio cholerae is an aquatic bacterium that inhabits coastal and estuarine areas, and is known to have several environmental reservoirs, including fish. Our laboratory has recently described the use of the zebrafish as a new animal model for the study of V. cholerae intestinal colonization, pathogenesis, and transmission.As early as six hours after exposure to V. cholerae, zebrafish develop diarrhea. Prior work in our laboratory has shown that this is not due to the action of cholera toxin. We hypothesize that accessory toxins produced by V. cholerae are the cause of diarrhea in infected zebrafish. In order to assess the effects of accessory toxins in the zebrafish, it was necessary to develop a method of quantifying diarrheal volume as a measure of pathogenesis. Here, we have adapted cell density, protein, and mucin assays, along with enumeration of V. cholerae in the zebrafish intestinal tract and in the infection water, to achieve this goal. Combined, these assays should help us determine which toxins have the greatest diarrheagenic effect in fish, and consequently, which toxins may play a role in environmental transmission.Importance Identification of the accessory toxins that cause diarrhea in zebrafish can help us understand more about the role of fish in the wild as aquatic reservoirs for V. cholerae It is plausible that accessory toxins can act to prolong colonization and subsequent shedding of V. cholerae back into the environment, thus perpetuating and facilitating transmission during an outbreak. It is also possible that accessory toxins help to maintain low levels of intestinal colonization in fish, giving V. cholerae an advantage when environmental conditions are not optimal for survival in the water. Studies such as this one are critical because fish could be an overlooked source of cholera transmission in the environment.

PMID: 28625997 [PubMed - as supplied by publisher]




Natural DNA transformation is functional in Lactococcus lactis ssp. cremoris KW2.
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Natural DNA transformation is functional in Lactococcus lactis ssp. cremoris KW2.

Appl Environ Microbiol. 2017 Jun 16;:

Authors: David B, Radziejwoski A, Toussaint F, Fontaine L, Henry de Frahan M, Patout C, van Dillen S, Boyaval P, Horvath P, Fremaux C, Hols P

Abstract
Lactococcus lactis is one of the most commonly used lactic acid bacteria in the dairy industry. Activation of competence for natural DNA transformation in this species would greatly improve the selection of novel strains with desired genetic traits. Here, we investigated the activation of natural transformation in L. lactis ssp. cremoris KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of proteins usually required for natural transformation. In the absence of knowledge about competence regulation in this species, we constitutively overproduced ComX in a reporter strain of late competence phase activation and showed, by transcriptomic analyses, a ComX-dependent induction of all key competence genes. We further demonstrated that natural DNA transformation is functional in this strain and requires the competence DNA uptake machinery. Since constitutive ComX overproduction is unstable, we alternatively expressed comX under the control of an endogenous xylose-inducible promoter. This regulated system was used to successfully inactivate the adaptor protein MecA and subunits of the Clp proteolytic complex, which were previously shown to be involved in ComX degradation in streptococci. In the presence of a low amount of ComX, the deletion of mecA, clpC, or clpP genes markedly increased the activation of the late competence phase and transformability. Altogether, our results report the functionality of natural DNA transformation in L. lactis and pave the way for the identification of signaling mechanisms that trigger the competence state in this species.IMPORTANCELactococcus lactis is a lactic acid bacterium of major importance, which is used as a starter species for milk fermentation, a host for heterologous protein production, and a delivery platform for therapeutic molecules. Here, we report the functionality of natural transformation in L. lactis ssp. cremoris KW2 by the overproduction of the master competence regulator ComX. The developed procedure enables a flexible approach to modify the chromosome with single point mutation, sequence insertion, or sequence replacement. These results represent an important step for the genetic engineering of L. lactis that will facilitate the design of strains optimized for industrial applications. This will also help to discover natural regulatory mechanisms controlling competence in the genus Lactococcus.

PMID: 28625996 [PubMed - as supplied by publisher]




NOVEL ANTIBIOTIC RESISTANCE DETERMINANTS FROM AGRICULTURAL SOIL EXPOSED TO ANTIBIOTICS WIDELY USED IN HUMAN MEDICINE AND ANIMAL FARMING.
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NOVEL ANTIBIOTIC RESISTANCE DETERMINANTS FROM AGRICULTURAL SOIL EXPOSED TO ANTIBIOTICS WIDELY USED IN HUMAN MEDICINE AND ANIMAL FARMING.

Appl Environ Microbiol. 2017 Jun 16;:

Authors: Lau CH, van Engelen K, Gordon S, Renaud J, Topp E

Abstract
Antibiotic resistance has emerged globally as one of the biggest threats to human and animal health. Although the excessive use of antibiotics is recognized for accelerating the selection for resistance, there is a growing body of evidence suggesting that natural environments are "hotspots" for the development of both ancient and contemporary resistance mechanisms. Given that pharmaceuticals can be entrained onto agricultural land through anthropogenic activities, this could be a potential driver for the emergence and dissemination of resistance in soil bacteria. Using functional metagenomics, we interrogated the "resistome" of bacterial communities found in a collection of Canadian agricultural soil, some of which had been receiving antibiotics widely used in human medicine (macrolides) or food animal production (sulfamethazine, chlortetracycline and tylosin) for up to 16 years. Of the 34 new antibiotic resistance genes (ARGs) recovered, the majority were predicted to encode for (multi)drug efflux systems, while a few share little to no homology with established resistance determinants. We characterized several novel gene products, including putative enzymes that can confer high-level resistance against aminoglycosides, sulfonamides, and broad range of beta-lactams, with respect to their resistance mechanisms and clinical significance. By coupling high-resolution proteomics analysis with functional metagenomics, we discovered an unusual peptide, PPP(AZI 4), encoded within an alternative open-reading frame not predicted by bioinformatics tools. Expression of the proline-rich PPP(AZI 4) can promote resistance against different macrolides but not other ribosomal-targeting antibiotics, implicating a new macrolide-specific resistance mechanism that could be fundamentally linked to the evolutionary design of this peptide.IMPORTANCE Antibiotic resistance is a clinical phenomenon with an evolutionary link to the microbial pangenome. Genes and protogenes encoding for specialized and potential resistance mechanisms are abundant in natural environments, but understanding of their identity and genomic context remain limited. Our discovery of several previously-unknown antibiotic resistance genes from uncultured soil microorganisms indicates that soil is a significant reservoir of resistance determinants, which, once acquired and "re-purposed" by pathogenic bacteria, can have serious impacts on therapeutic outcomes. This study provides valuable insights into the diversity and identity of resistance within the soil microbiome. The finding of a novel peptide-mediated resistance mechanism involving an unpredicted gene product also highlights the usefulness of integrating proteomics analysis into metagenomics-driven gene discovery.

PMID: 28625995 [PubMed - as supplied by publisher]




A Small Protein, HemP, is a Transcriptional Activator for Hemin Uptake Operon in Burkholderia multivorans ATCC 17616.
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A Small Protein, HemP, is a Transcriptional Activator for Hemin Uptake Operon in Burkholderia multivorans ATCC 17616.

Appl Environ Microbiol. 2017 Jun 16;:

Authors: Sato T, Nonoyama S, Kimura A, Nagata Y, Ohtsubo Y, Tsuda M

Abstract
Iron and heme play very important roles in various metabolic functions in bacteria, and their intracellular homeostasis is maintained because high concentrations of free forms of these molecules greatly facilitate the Fenton reaction-mediated production of large amounts of reactive oxygen species that severely damage various biomolecules. Ferric uptake regulator (Fur) from Burkholderiamultivorans ATCC 17616 is an iron-responsive global transcriptional regulator, and its fur deletant exhibits pleiotropic phenotypes. In this study, we found that the phenotypes of the fur deletant were suppressed by an additional mutation in hemP The hemP transcription was negatively regulated by Fur under the iron-replete conditions, and was constitutive in the fur deletant. Growth of a hemP deletant was severely impaired in a medium containing hemin as a sole iron source, demonstrating the important role of HemP in the hemin utilization. HemP was required as a transcriptional activator that specifically binds the promoter-containing region upstream of a Fur-repressive hmuRSTUV operon, which encodes the proteins for hemin uptake. A hmuR deletant was still able to grow using hemin as a sole iron source, albeit at a rate clearly slower than that of the wild-type strain. These results strongly suggested (i) the involvement of HmuR in hemin uptake and (ii) the presence in ATCC 17616 of at least a part of an(other) unknown hemin-uptake system(s) whose expression depends on the HemP function. Our in vitro analysis also indicated high-affinity binding of HemP to hemin, and such a property might modulate the transcriptional activation of the hmu operon.IMPORTANCE Although the hmuRSTUV genes for the utilization of hemin as a sole iron source have been identified in a few Burkholderia strains, the regulatory expression of these genes has remained unknown. Our analysis in this study using B. multivorans ATCC 17616 showed that its HemP protein is required for the expression of the hmuRSTUV operon, and the role of HemP in β-proteobacterial species was elucidated for the first time in this study. The HemP protein was also found to have two additional properties that have not been reported for the functional homologues in other species; one is that that HemP is able to bind to the promoter-containing region of the hmu operon so as to directly activate its transcription, and the other is that HemP is also required for the expression of an unknown hemin-uptake system.

PMID: 28625994 [PubMed - as supplied by publisher]




Survival of H5N1 Highly Pathogenic Avian Influenza Virus in Tissues Derived from Experimentally Infected Chickens.
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Survival of H5N1 Highly Pathogenic Avian Influenza Virus in Tissues Derived from Experimentally Infected Chickens.

Appl Environ Microbiol. 2017 Jun 16;:

Authors: Yamamoto Y, Nakamura K, Mase M

Abstract
Eurasian lineage H5N1 highly pathogenic avian influenza (HPAI) virus has been a severe threat to the poultry industry since its emergence in 1996. The carcass or tissues derived from infected birds may have the risk of the virus spreading to humans, animals, and the surrounding environment. In this study, we investigated the survival of the virus in feather, muscle, and liver tissues collected from six chickens (Gallus gallus) experimentally infected with H5N1 HPAI virus. The tissues were stored at +4°C or +20°C and viral isolation was performed at different times for 360 days. The maximum period for viral survival was observed in samples stored at +4°C in all tissue types, i.e., 240 days in feather tissues, 160 days in muscle, and 20 days in liver. The viral infectivity at +20°C was maintained for a maximum of 30 days in the feather tissues, 20 days in muscle, and 3 days in liver. The viral inactivation rates partly overlapped in the feather and muscle tissues at both temperatures. The virus was inactivated rapidly in the liver. Our experimental results indicate that the tissue type and temperature can greatly influence the survival of H5N1 HPAI virus in the tissues of infected chickens.IMPORTANCE H5N1 subtype highly pathogenic avian influenza virus can cause massive losses of poultry and people need to handle a large number of chicken carcasses contaminated with the virus at the outbreak sites. This study evaluated how long the virus can keep its infectivity in the three types of tissues derived from chickens infected with the virus. Our experimental results indicate that the virus can survive in tissues for a specific period of time depending on the tissue type and temperature. Our results are valuable for better understanding viral ecology in the environment, and for reducing the risk of the virus spreading via bird tissues contaminated with the virus.

PMID: 28625993 [PubMed - as supplied by publisher]




Flow cytometry, a powerful novel tool to assess viability of fungal conidia in metal working fluids.
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Flow cytometry, a powerful novel tool to assess viability of fungal conidia in metal working fluids.

Appl Environ Microbiol. 2017 Jun 16;:

Authors: Vanhauteghem D, Demeyere K, Callaert N, Boelaert A, Haesaert G, Audenaert K, Meyer E

Abstract
Fungal contamination of metal working fluids (MWF) is a dual problem in automatized processing plants because resulting fungal biofilms obstruct cutting, drilling and polishing machines. Moreover, some fungal species of MWF comprise pathogens such as Fusarium solani Therefore, the development of an accurate analytical tool to evaluate conidia viability in MWF is important. We developed a flow cytometric method to measure fungal viability in MWF using F. solani as model organism. To validate this method, viable and dead conidia were mixed in several proportions and flow cytometrically analyzed. Subsequently, we assessed the fungicidal activity of two commercial MWF using flow cytometry (FCM) and compared it with microscopic analyses and plating experiments. We evaluated the fungal growth in both MWF after 7 days using qPCR to assess the predictive value of FCM. Our results show that FCM distinguishes live from dead conidia as early as 5 hours after exposure to MWF whereas the microscopic germination approach detected conidial viability much later and less accurately. At 24h, microscopic analyses of germinating conidia and live/dead analyses by FCM correlated well although the former consistently underestimated the proportion of viable conidia. In addition, reproducibility and sensitivity of the flow cytometric method was high and allowed to assess the fungicidal properties of two commercial MWF. Importantly, the obtained flow cytometric results on viability of F. solani conidia at both early time points (5h and 24h) correlated well with fungal biomass measurements assessed via a qPCR methodology 7 d after the start of the experiment.Importance This result depicts the predictive power of FCM in assessing the fungicidal capacity of MWF formulations. It also implies that FCM can be implemented as a rapid detection tool to estimate the viable fungal load in an industrial processing matrix (MWF).

PMID: 28625992 [PubMed - as supplied by publisher]




Different amplicon targets for sequencing-based studies of fungal diversity.
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Different amplicon targets for sequencing-based studies of fungal diversity.

Appl Environ Microbiol. 2017 Jun 16;:

Authors: De Filippis F, Laiola M, Blaiotta G, Ercolini D

Abstract
Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes with the 16S rRNA being the gold standard for bacteria. As for Fungi, the most commonly used target is the internal transcribed spacer (ITS). However, the uneven ITS length among species may promote preferential amplification and sequencing and incorrect estimation of their abundance. Therefore, the use of different targets is desirable. We evaluated the use of three different target amplicons for the characterization of fungal diversity. After an in silico primer evaluation, we compared three amplicons (ITS 1-2, 18S ribosomal small subunit RNA and D1/D2 domain of the 26S ribosomal large subunit RNA) both using biological samples and a mock community of common fungal species. All the three targets allowed an accurate identification of the species present. Nevertheless, high heterogeneity in ITS 1-2 length was found and this caused an over-estimation of the abundance of species with shorter ITS, while both 18S and 26S amplicons allowed a more reliable quantification. We demonstrated that ITS 1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities and efforts should be done for promoting the use of different targets in sequencing-based microbial ecology studies.Importance Amplicon-sequencing approaches for fungi may rely on different targets affecting diversity and abundance of the fungal species. An increasing number of studies will address the fungal diversity by high-throughput amplicon sequencing. The description of the communities must be accurate and reliable in order to draw useful insights and to address both ecological and biological questions. By analysing a mock community and several biological samples we demonstrate that using different amplicon targets may change the results of fungal microbiota composition and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities.

PMID: 28625991 [PubMed - as supplied by publisher]




Bioavailability of carbohydrate content in natural and transgenic switchgrasses for the extreme thermophile Caldicellulosiruptor bescii.
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Bioavailability of carbohydrate content in natural and transgenic switchgrasses for the extreme thermophile Caldicellulosiruptor bescii.

Appl Environ Microbiol. 2017 Jun 16;:

Authors: Zurawski JV, Khatibi PA, Akinosho HO, Straub CT, Compton SH, Conway JM, Lee LL, Ragauskas AJ, Davison BH, Adams MWW, Kelly RM

Abstract
Improving access to the carbohydrate content of lignocellulose is key to reducing recalcitrance for microbial deconstruction and conversion to fuels and chemicals. Caldicellulosiruptor bescii completely solubilizes naked microcrystalline cellulose, yet this transformation is impeded within the context of the plant cell wall by a network of lignin and hemicellulose. Here, the bioavailability of carbohydrates to C. bescii at 70°C was examined for reduced lignin transgenic switchgrass lines COMT3(+) and MYB Trans, their corresponding parental lines (cultivar Alamo) COMT3(-) and MYB WT, and natural variant cultivar Cave-in-Rock (CR). Transgenic modification improved carbohydrate solubilization by C. bescii to 15% (2.3-fold) for MYB, and to 36% (1.5-fold) for COMT, comparable to the levels achieved for the natural variant, CR (36%). Carbohydrate solubilization was nearly doubled after two consecutive microbial fermentations compared to one microbial step, but never exceeded 50% overall. Hydrothermal treatment (180°C) prior to microbial steps improved solubilization 3.7-fold for the most recalcitrant line (MYB WT), and increased carbohydrate recovery to nearly 50% for the least recalcitrant lines (COMT3(+) and CR). Alternating microbial and hydrothermal steps (T→M→T→M) further increased bioavailability, achieving carbohydrate solubilization ranging from 50% for MYB WT to above 70% for COMT3(-), COMT3(+) and CR. Incomplete carbohydrate solubilization suggests that cellulose in the highly lignified residue was inaccessible; indeed, residue from T→M→T→M treatment was primarily glucan and inerts (lignin and ash). While C. bescii could significantly solubilize the transgenic switchgrass lines and natural variant tested here, additional or alternative strategies (physical, chemical, enzymatic, and/or genetic) are needed to eliminate recalcitrance.IMPORTANCE Key to a microbial process for solubilization of plant biomass is the organism's access to the carbohydrate content of lignocellulose. Economically viable routes will characteristically minimize physical, chemical and biological pretreatment such that microbial steps contribute to the greatest extent possible. Recently, transgenic versions of plants and trees have been developed with the intention of lowering the barrier to lignocellulose conversion, with particular focus on lignin content and composition. Here, the extremely thermophilic bacterium, Caldicellulosiruptor bescii, was used to solubilize natural and genetically modified switchgrass lines, with and without the aid of hydrothermal treatment. For lignocellulose conversion, it is clear that the integration of microorganism, plant biomass substrate and processing steps must all be considered simultaneously to achieve optimal results. Whether switchgrass lines engineered for low lignin or natural variants with desirable properties are used, conversion will depend on microbial access to crystalline cellulose in the plant cell wall.

PMID: 28625990 [PubMed - as supplied by publisher]




Rapid, high-throughput identification of anthrax-causing and emetic Bacillus cereus group genome assemblies using BTyper, a computational tool for virulence-based classification of Bacillus cereus group isolates using nucleotide sequencing data.
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Rapid, high-throughput identification of anthrax-causing and emetic Bacillus cereus group genome assemblies using BTyper, a computational tool for virulence-based classification of Bacillus cereus group isolates using nucleotide sequencing data.

Appl Environ Microbiol. 2017 Jun 16;:

Authors: Carroll LM, Kovac J, Miller RA, Wiedmann M

Abstract
The Bacillus cereus group comprises nine species, several of which are pathogenic. Differentiating between isolates that may cause disease and those that do not is a matter of public health and economic importance, but can be particularly challenging due to the high genomic similarity of the group. To this end, we have developed BTyper, a computational tool that employs a combination of (i) virulence gene-based typing, (ii) multi-locus sequence typing (MLST), (iii) panC clade typing, and (iv) rpoB allelic typing to rapidly classify B. cereus group isolates using nucleotide sequencing data. BTyper was applied to a set of 662 B. cereus group genome assemblies to (i) identify anthrax-associated genes in non-B. anthracis members of the B. cereus group, and (iI) identify assemblies from B. cereus group strains with emetic potential. With BTyper, anthrax toxin genes cya, lef and pagA were detected in 8 genomes classified in NCBI as B. cereus that clustered into two distinct groups using k-medoids clustering, while B. anthracis poly-γ-D-glutamate capsule biosynthesis genes capABCDE or hyaluronic acid capsule gene hasA were detected in an additional 16 assemblies classified as either B. cereus or B. thuringiensis isolated from clinical, environmental, and food sources. Emetic toxin genes cesABCD were detected in 24 assemblies belonging to panC clades III and VI that had been isolated from food, clinical, and environmental settings. The command line version of BTyper is available at https://github.com/lmc297/BTyper In addition, BMiner, a companion application for analyzing multiple BTyper output files in aggregate, can be found at https://github.com/lmc297/BMinerImportanceBacillus cereus is a foodborne pathogen that is estimated to cause tens of thousands of illnesses each year in the United States alone. Even with molecular methods, it can be difficult to distinguish non-pathogenic B. cereus group isolates from their pathogenic counterparts, including the human pathogen B. anthracis, which is responsible for anthrax, as well as insect pathogen B. thuringiensis By using the variety of typing schemes employed by BTyper, users can rapidly classify, characterize, and assess the virulence potential of any isolate using its nucleotide sequencing data.

PMID: 28625989 [PubMed - as supplied by publisher]




Modification of a pollen trap design to capture airborne conidia of Entomophaga maimaiga and detection by quantitative PCR.
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Modification of a pollen trap design to capture airborne conidia of Entomophaga maimaiga and detection by quantitative PCR.

Appl Environ Microbiol. 2017 Jun 16;:

Authors: Bittner TD, Hajek AE, Liebhold AM, Thistle H

Abstract
The goal of this study was to develop effective and practical field sampling methods for quantifying aerial deposition of airborne conidia of Entomophaga maimaiga over space and time. This important fungal pathogen is a major cause of larval mortality in invasive gypsy moth (Lymantria dispar) populations in the United States. Airborne conidia of this pathogen are relatively large (similar in size to pollen) with unusual characteristics, requiring specialized methods for collection and quantification. Initially, dry sampling (settling of spores from the air onto a dry surface) was used to confirm the detectability of E. maimaiga at field sites with L. dispar mortality caused by E. maimaiga using quantitative PCR (qPCR) methods. We then measured the signal degradation of conidial DNA on dry surfaces under field conditions, ultimately rejecting dry sampling as a reliable method due to rapid DNA degradation. We modified a chamber-style trap commonly used in palynology to capture settling spores in buffer. We tested this wet-trapping method in a large-scale (137 km) spore-trapping survey across gypsy moth outbreak regions undergoing epizootics in Pennsylvania in the summer of 2016. Using four-day collection periods during the period of late instar and pupal development, we detected variable amounts of target DNA settling from the air. The amounts declined over the season and with distance from the nearest defoliated area, indicating airborne spore dispersal from outbreak areas.IMPORTANCE We report on a method for trapping and quantifying airborne spores of Entomophaga maimaiga, an important fungal pathogen impacting gypsy moth (Lymantria dispar) populations. This method can be used to track dispersal of E. maimaiga from epizootic areas and ultimately provide critical understanding of the spatial dynamics of gypsy moth-pathogen interactions.

PMID: 28625988 [PubMed - as supplied by publisher]




Metabolic engineering of Actinobacillus succinogenes provides insights into succinic acid biosynthesis.
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Metabolic engineering of Actinobacillus succinogenes provides insights into succinic acid biosynthesis.

Appl Environ Microbiol. 2017 Jun 16;:

Authors: Guarnieri MT, Chou YC, Salvachúa D, Mohagheghi A, St John PC, Peterson DJ, Bomble YJ, Beckham GT

Abstract
Actinobacillus succinogenes, a gram-negative, facultative anaerobe, exhibits the native capacity to convert pentose and hexose sugars to succinic acid (SA) with high yield as a tricarboxylic acid (TCA) cycle intermediate. In addition, A. succinogenes is capnophilic, incorporating CO2 into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO2 to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in A. succinogenes, enabling examination of SA flux determinants via knockout of the primary competing pathways - namely acetate and formate production - and overexpression of the key enzymes in the reductive branch of the TCA cycle leading to SA. Batch fermentation experiments with the wild-type and engineered strains using pentose-rich sugar streams demonstrate that the overexpression of the SA biosynthetic machinery, in particular the enzyme malate dehydrogenase, enhances flux to SA. Additionally, removal of competitive carbon pathways leads to higher purity SA, but also triggers the generation of byproducts not previously described in this organism (e.g., lactic acid). The resultant engineered strains also lend insight into energetic and redox balance and elucidate mechanisms governing organic acid biosynthesis in this important natural SA-producing microbe.IMPORTANCE Succinic acid production from lignocellulosic residues is a potential route to enhance the economic feasibility of modern biorefineries. Herein, we have employed facile genetic tools to systematically manipulate competing acid production pathways and overexpress the succinic acid-producing machinery in Actinobacillus succinogenes Furthermore, the resulting strains are evaluated via fermentation on relevant pentose-rich sugar streams from corn stover. Overall, this work demonstrates genetic modifications that can lead to succinic acid production improvements, and also identifies key flux determinants and new bottlenecks and energetic needs when removing by-product pathways in A. succinogenes metabolism.

PMID: 28625987 [PubMed - as supplied by publisher]




The Irr and RirA proteins participate in a complex regulatory circuit and act in concert to modulate bacterioferritin expression in Ensifer meliloti 1021.
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The Irr and RirA proteins participate in a complex regulatory circuit and act in concert to modulate bacterioferritin expression in Ensifer meliloti 1021.

Appl Environ Microbiol. 2017 Jun 16;:

Authors: Costa D, Amarelle V, Valverde C, O'Brian MR, Fabiano E

Abstract
In this work we found that the bfr gene of the rhizobial species Ensifer meliloti, encoding a bacterioferritin iron storage protein, is involved in iron homeostasis and oxidative stress response. This gene is located downstream and overlapping the smc03787 ORF. No well predicted RirA or Irr boxes were found in the region immediately upstream the bfr gene although two presumptive RirA boxes and one presumptive Irr box were present in the putative promoter of smc03787 We demonstrate that bfr gene expression is enhanced under iron sufficient condition and that Irr and RirA modulate this expression. The pattern of bfr gene expression as well as the response to Irr and RirA, are inversely correlated to that of smc03787 Moreover, our results suggest that the small RNA SmelC759 participates in RirA- and Irr-mediated regulation of bfr-expression, and that additional unknown factors are involved in iron-dependent regulationImportanceE. meliloti belongs to the Alphaproteobacteria, a group of bacteria that includes several relevant species able to associate with eukaryotic hosts, from mammals to plants, in a symbiotic or pathogenic manner. Regulation of iron homeostasis in this group of bacteria differs from that found in the well-studied Gammaproteobacteria. In this work we analyzed the effect of rirA and irr mutations in bfr gene expression. We demonstrate the effect of an irr mutation in iron homeostasis in this bacterial genus. Moreover, results obtained indicate a complex regulatory circuit where multiple regulators, including RirA, Irr, the small RNA SmelC759 and still unknown factors, act in concert to balance bfr gene expression.

PMID: 28625986 [PubMed - as supplied by publisher]




Periplasmic Nicotine Dehydrogenase NdhAB Utilizes Pseudoazurin as Its Physiological Electron Acceptor in Agrobacterium tumefaciens S33.
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Periplasmic Nicotine Dehydrogenase NdhAB Utilizes Pseudoazurin as Its Physiological Electron Acceptor in Agrobacterium tumefaciens S33.

Appl Environ Microbiol. 2017 Jun 16;:

Authors: Yu W, Wang R, Huang H, Xie H, Wang S

Abstract
Agrobacterium tumefaciens S33 can grow with nicotine as the sole source of carbon, nitrogen, and energy via a novel hybrid of the pyridine pathway and the pyrollidine pathway. Characterization of the enzymes involved in the hybrid pathway is important for understanding its biochemical mechanism. Here we report that the molybdenum-containing nicotine dehydrogenase (NdhAB), which catalyzes the initial step of nicotine degradation, is located in the periplasm of strain S33, while the 6-hydroxynicotine oxidase and 6-hydroxypseudooxynicoine oxidase are in the cytoplasm. This is consistent with the fact that NdhA has a Tat signal peptide. Interestingly, an ORF adjacent to the ndhAB gene was verified to encode a copper-containing electron carrier, pseudoazurin (Paz), that has a typical signal peptide of bacterial Paz. Both were transported into the periplasm after being produced in the cytoplasm. We purified NdhAB from the periplasmic fraction of strain S33 and found that, with Paz as the physiological electron acceptor, NdhAB catalyzed the hydroxylation of nicotine at a specific rate of 110.52 ± 8.09 μmol min(-1) mg protein(-1), where the oxygen atom in the hydroxyl group of the product 6-hydroxynicotine was derived from H2O. The apparent Km values for nicotine and Paz were 1.64 ± 0.07 μM and 3.61 ± 0.23 μM, respectively. NAD(P)(+), O2, and ferredoxin could not serve as electron acceptors. Disruption of the paz gene disabled the strain for nicotine degradation, indicating that Paz is required for nicotine catabolism in the strain. These findings help our understanding of electron transfer during nicotine degradation in bacteria.IMPORTANCE Nicotine is a toxic and addictive N-heterocyclic aromatic alkaloid produced in tobacco. Its catabolism in organisms and degradation in tobacco wastes have become the major concerns of health and the environment. Bacteria usually decompose nicotine using the classical strategy of hydroxylating the pyridine ring with the help of activated oxygen by nicotine dehydrogenase, which binds one molybdopterin, two [lsqb]2Fe2S[rsqb] clusters, and usually one FAD as well. However, the physiological electron acceptor for the reaction is still unknown. In this study, we found the two-component nicotine dehydrogenase from Agrobacterium tumefaciens S33, naturally lacking FAD-binding domain, is located in the periplasmic space and uses a copper-containing electron carrier, pseudoazurin, as its physiological electron acceptor. We report here the role of pseudoazurin in a reaction catalyzed by a molybdopterin-containing hydroxylase occurring in the periplasmic space. These results provide new biochemical knowledge in microbial degradation of N-heterocyclic aromatic compound.

PMID: 28625985 [PubMed - as supplied by publisher]




Indole-induced Reversion of Intrinsic Multi-antibiotic Resistance in Lysobacter enzymogenes.
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Indole-induced Reversion of Intrinsic Multi-antibiotic Resistance in Lysobacter enzymogenes.

Appl Environ Microbiol. 2017 Jun 16;:

Authors: Han Y, Wang Y, Yu Y, Chen H, Shen Y, Du L

Abstract
Lysobacter are a group of environmental bacteria that are emerging as a new source of antibiotics. One characteristic of Lysobacter is intrinsic resistance to multiple antibiotics, which had not been studied. To understand the resistant mechanism, we tested the effect of blocking two-component regulator systems (TCS) on antibiotic resistance of L. enzymogenes, a prolific producer of antibiotics. Upon treatment with LED209, an inhibitor of the widespread TCS QseC/QseB, L. enzymogenes produced a large amount of an unknown metabolite that was barely detectable in the untreated culture. Subsequent structural elucidation by NMR unexpectedly revealed the metabolite to be indole. Indole production was also markedly induced by adrenaline, a known modulator of QseC/QseB. Next, we identified two TCS genes, Le-qseC/Le-qseB, in L. enzymogenes and found that mutants of Le-qseC/Le-qseB also led to a dramatic increase of indole production. We then chemically synthesized a fluorescent indole probe that could label the cells. While mutant Le-qseB (cytoplasmic response regulator) could be clearly labeled by the probe, mutant Le-qseC (membrane sensor) was not labeled. It had been reported that indole could enhance antibiotic resistance in bacteria. Therefore, we tested if the dramatic increase of indole production in L. enzymogenes upon blocking Le-qseC/Le-qseB would lead to an enhanced antibiotic resistance. Surprisingly, we found that indole caused the intrinsically multi-antibiotic resistant L. enzymogenes to become susceptible. Point mutations at conserved amino acids in Le-QseC also led to antibiotic susceptibility. Because indole is known as an interspecies signal, the findings may have implications.IMPORTANCE The environmental bacteria Lysobacter are a new source of antibiotic compounds and exhibit intrinsic antibiotic resistance. Here, we found that inactivation of a two-component regulator system (TCS) by inhibitor or by gene deletion led to a remarkable increase of a metabolite's production in L. enzymogenes, and this metabolite was identified to be indole. We chemically synthesized a fluorescent indole probe and found that it could label the wild type and mutant of the TCS' cytoplasmic response regulator, but not mutant of the TCS' membrane sensor. Indole treatment reversed the intrinsically multi-drug resistant L. enzymogenes to be susceptible to antibiotics. Mutations of the TCS sensor also led to antibiotic susceptibility. Because indole is known as an interspecies signal between gut microbiota and mammalian hosts, the observation that indole could render the intrinsically resistant L. enzymogenes susceptible to common antibiotics may have implications.

PMID: 28625984 [PubMed - as supplied by publisher]




Strain-level metagenomic analysis of the fermented dairy beverage nunu highlights potential food safety risks.
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Strain-level metagenomic analysis of the fermented dairy beverage nunu highlights potential food safety risks.

Appl Environ Microbiol. 2017 Jun 16;:

Authors: Walsh AM, Crispie F, Daari K, O'Sullivan O, Martin JC, Arthur CT, Claesson MJ, Scott KP, Cotter PD

Abstract
The rapid detection of pathogenic strains in food products is essential for the prevention of disease outbreaks. It has already been demonstrated that whole metagenome shotgun sequencing can be used to detect pathogens in food but, until recently, strain-level detection of pathogens has relied on whole metagenome assembly, which is a computationally demanding process. Here, we demonstrate that three short read alignment-based methods, MetaMLST, PanPhlAn, and StrainPhlAn, can accurately, and rapidly, identify pathogenic strains in spinach metagenomes which were intentionally spiked with Shiga toxin-producing Escherichia coli in a previous study. Subsequently, we employ the methods, in combination with other metagenomics approaches, to assess the safety of nunu, a traditional Ghanaian fermented milk product which is produced by the spontaneous fermentation of raw cow milk. We show that nunu samples are frequently contaminated with bacteria associated with the bovine gut, and worryingly, we detect putatively pathogenic E. coli and Klebsiella pneumoniae strains in a subset of nunu samples. Ultimately, our work establishes that short read alignment-based bioinformatics approaches are suitable food safety tools, and we describe a real-life example of their utilisation.Importance Foodborne pathogens are responsible for millions of illnesses, annually. Here, we demonstrate that short read alignment-based bioinformatics tools can accurately, and rapidly, detect pathogenic strains in food products from shotgun metagenomics data. The methods used here are considerably faster than both traditional culturing methods and alternative bioinformatics approaches that rely on metagenome assembly, and thus they can potentially be used for more high-throughput food safety testing. Overall, our results suggest that whole metagenome sequencing can be used as a practical food safety tool to prevent diseases or link outbreaks to specific food products.

PMID: 28625983 [PubMed - as supplied by publisher]




The novel stress survival islet 2 (SSI-2), predominantly present in Listeria monocytogenes strains of ST121, is involved in alkaline and oxidative stress response.
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The novel stress survival islet 2 (SSI-2), predominantly present in Listeria monocytogenes strains of ST121, is involved in alkaline and oxidative stress response.

Appl Environ Microbiol. 2017 Jun 16;:

Authors: Harter E, Wagner EM, Zaiser A, Halecker S, Wagner M, Rychli K

Abstract
The food-borne pathogen Listeria (L) monocytogenes is able to survive a variety of stress conditions leading to the colonization of different niches like the food processing environment. This study focuses on the hypervariable genetic hotspot lmo0443-lmo0449 haboring three inserts: the stress survival islet 1 (SSI-1), the single-gene insert LMOf2365_0481 and two homologous genes of the non-pathogenic species L. innocua: lin0464, a putative transcriptional regulator and lin0465, an intracellular PfpI protease. Our prevalence study revealed a different distribution of the inserts between human and food-associated isolates. The lin0464-lin0465 insert was predominantly found in food-associated strains of sequence type (ST) 121. Functional characterization of this insert showed that the putative PfpI protease Lin0465 is involved in alkaline and oxidative stress response, but not in acidic, gastric, heat, cold, osmotic and antibiotic stress. In parallel, deletion of lin0464 decreased the survival under alkaline and oxidative stress. The expression of both genes increased significantly under oxidative stress conditions independently of the alternative sigma factor σ(B) Furthermore, we showed that the expression of the protease lin0465 is regulated by the transcription factor lin0464 under stress conditions, suggesting that lin0464 and lin0465 form a functional unit.In conclusion, we identified a novel stress survival islet 2 (SSI-2), predominantly present in L. monocytogenes ST121 strains, beneficial for survival under alkaline and oxidative stress, potentially supporting adaptation and persistence of L. monocytogenes in food processing environments.IMPORTANCEListeria (L.) monocytogenes strains of ST121 are known to persist for months and even years in food processing environments, thereby increasing the risk of food contamination and listeriosis. However, the molecular mechanism underlying this remarkable niche-specific adaptation is still unknown. Here, we demonstrate that the genomic islet SSI-2, predominantly present in L. monocytogenes ST121 strains, is beneficial for survival under alkaline and oxidative stress conditions, which are routinely encountered in food processing environments. Our findings suggest that SSI-2 is part of a diverse set of molecular determinants contributing to niche-specific adaptation and persistence of L. monocytogenes ST121 strains in food processing environments.

PMID: 28625982 [PubMed - as supplied by publisher]




Heterologous expression of the Clostridium carboxidivorans CO dehydrogenase alone or together with the acetyl-CoA synthase enables Clostridium acetobutylicum to both reduce CO2 and oxidize CO.
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Heterologous expression of the Clostridium carboxidivorans CO dehydrogenase alone or together with the acetyl-CoA synthase enables Clostridium acetobutylicum to both reduce CO2 and oxidize CO.

Appl Environ Microbiol. 2017 Jun 16;:

Authors: Carlson ED, Papoutsakis ET

Abstract
With recent advances in synthetic biology, CO2 could be utilized as a carbon feedstock by native or engineered organisms, assuming the availability of electrons. Two key enzymes used in autotrophic CO2 fixation are the CO dehydrogenase (CODH) and acetyl-CoA synthase (ACS), which form a bifunctional heterotetrameric complex. CODH/ACS can reversibly catalyze CO2 to CO, effectively enabling a biological water-gas shift reaction at ambient temperatures and pressures. The CODH/ACS complex is part of the Wood-Ljungdahl pathway (WLP) used by acetogens to fix CO2 and has been well characterized in native hosts. So far, only a few recombinant CODH/ACS complexes have been expressed in heterologous hosts, none of which demonstrated in vivo CO2 reduction. Here, functional expression of the Clostridium carboxidivorans CODH/ACS complex is demonstrated in the solventogen C. acetobutylicum, which was engineered to express CODH alone or together with the ACS. Both strains exhibited CO2 reduction and CO oxidation activities. The CODH reactions were interrogated using isotopic labeling, thus verifying that CO was a direct product of CO2 reduction and vice versa. CODH apparently uses a native C. acetobutylicum ferredoxin as an electron carrier for CO2 reduction. Heterologous CODH activity depended on actively growing cells and required the addition of nickel, which is inserted into CODH without the need to express the native Ni insertase protein. Increasing CO concentrations in the gas phase inhibited CODH activity and altered the metabolite profile of the CODH-expressing cells. This work provides the foundation for engineering a complete and functional WLP in non-native host organisms.IMPORTANCE Functional expression of CO dehydrogenase (CODH) from Clostridium carboxidivorans was demonstrated in C. acetobutylicum, which is natively incapable of CO2 fixation. Expression of CODH, alone or together with the C. carboxidivorans acetyl-CoA synthase (ACS), enabled C. acetobutylicum to catalyze both CO2 reduction and CO oxidation. Importantly, CODH exhibited activity both in the presence and absence of ACS, which has never been shown before. (13)C-tracer studies confirmed that the engineered C. acetobutylicum strains can reduce CO2 to CO, and also oxidize CO, during growth on glucose.

PMID: 28625981 [PubMed - as supplied by publisher]




Functional analysis of a novel β-(1,3)-glucanase from Corallococcus sp. EGB containing a fascin-like module.
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Functional analysis of a novel β-(1,3)-glucanase from Corallococcus sp. EGB containing a fascin-like module.

Appl Environ Microbiol. 2017 Jun 16;:

Authors: Zhou J, Li Z, Wu J, Li L, Li D, Ye X, Luo X, Huang Y, Cui Z, Cao H

Abstract
A novel β-(1,3)-glucanase gene designated lamC, cloned from Corallococcus sp. EGB, contains a fascin-like module and a glycoside hydrolase family 16 (GH16) catalytic module. LamC displays broad hydrolytic activity toward various polysaccharides. Analysis of the hydrolytic products revealed that LamC is an exo-acting enzyme on β-(1,3)- and β-(1,6)-linked glucan substrates and an endo-acting enzyme on β-(1,4)-linked glucan and xylan substrates. Site-directed mutagenesis of conserved catalytic Glu residues (E304A and E309A) demonstrated that these activities were derived from the same active site. Excision of the fascin-like module resulted in decreased activity toward β-(1,3)-linked glucans. The carbohydrate-binding assay showed that the fascin-like module was a novel β-(1,3)-linked glucan-binding module. The functional characterization of the fascin-like module and catalytic module will help us better understand these enzymes and modules.IMPORTANCE In this report of a bacterial β-(1,3)-glucanase containing a fascin-like module, we reveal the β-(1,3)-glucan-binding function of the fascin-like module present in the N-terminus of LamC. LamC displays exo-β-(1,3)/(1,6)-glucanase and endo-β-(1,4)-glucanase/xylanase activities with a single catalytic domain. Thus, LamC was identified as a novel member of the GH16 family.

PMID: 28625980 [PubMed - as supplied by publisher]




Correction for Wertz et al., "Genomic and Physiological Characterization of the Verrucomicrobia Isolate Didymococcus colitermitum gen. nov., sp. nov., Reveals Microaerophily and Nitrogen Fixation Genes".
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Correction for Wertz et al., "Genomic and Physiological Characterization of the Verrucomicrobia Isolate Didymococcus colitermitum gen. nov., sp. nov., Reveals Microaerophily and Nitrogen Fixation Genes".

Appl Environ Microbiol. 2017 Jul 01;83(13):

Authors: Wertz JT, Kim E, Breznak JA, Schmidt TM, Rodrigues JLM

PMID: 28625969 [PubMed - in process]