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Experimental Manipulation Shows a Greater Influence of Population than Dietary Perturbation on the Microbiome of Tyrophagus putrescentiae.
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Experimental Manipulation Shows a Greater Influence of Population than Dietary Perturbation on the Microbiome of Tyrophagus putrescentiae.

Appl Environ Microbiol. 2017 Feb 24;:

Authors: Erban T, Ledvinka O, Nesvorna M, Hubert J

Abstract
Tyrophagus putrescentiae (TP) is inhabited by bacteria that differ among mite populations (strains) and diets. Here, we investigated how the microbiome and fitness of TP are altered by dietary perturbations and mite populations. Four TP populations, referred to as Dog, Koppert, Laboratory and Phillips, underwent a perturbation, i.e., a dietary switch from a rearing diet to two experimental diets. The microbiome was investigated by sequencing the V1-V3 portion of the 16S rRNA gene, and selected bacterial taxa were quantified by qPCR using group/taxa-specific primers. The parameters observed were the changes in mite population growth and nutritional status, i.e., the contents of total glycogen, lipids, saccharides, and proteins in mites. The effect of diet perturbation on the variability of the microbiome composition and population growth was lower than the effect induced by the mite populations. By contrast, the diet perturbation showed a greater effect on nutritional status of mites than the mite populations. The endosymbionts exhibited high variations among TP populations, including Cardinium in the Laboratory population, Blattabacterium-like in the Dog population, and Wolbachia in the Dog and Phillips populations. Solitalea-like and Bartonella-like were present in the Dog, Koppert and Phillips populations in different proportions. The TP microbiome is dynamic and differs based on both the mite population and perturbation; however, the mites remain characterized by robust bacterial communities. Bacterial endosymbionts were found in all populations but represented a dominant portion of the microbiome in only some populations.IMPORTANCE We addressed the question of whether population origin or perturbation exerts a more significant influence on the bacterial community of the stored product mite Tyrophagus putrescentiae (TP). The microbiomes of four populations of TP subjected to diet perturbation were compared. Based on our results, the bacterial community was more affected by the mite population than by diet perturbation. This result can be interpreted as a high stability of the putative intracellular symbionts to dietary perturbation. The changes in the absolute and relative numbers of Wolbachia, Blattabacterium-like, Solitalea-like and Cardinium in the TP populations can also be caused by neutral processes other than perturbation. When considering nutritional status, the effect of population appeared less important than the perturbation. We hypothesize that differences in proportions of the endosymbiotic bacteria result in changes in mite population growth.

PMID: 28235879 [PubMed - as supplied by publisher]




Botrytis fragariae, a new species causing gray mold on strawberries, shows high frequencies of specific and efflux-based fungicide resistance.
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Botrytis fragariae, a new species causing gray mold on strawberries, shows high frequencies of specific and efflux-based fungicide resistance.

Appl Environ Microbiol. 2017 Feb 24;:

Authors: Rupp S, Plesken C, Rumsey S, Dowling M, Schnabel G, Weber RW, Hahn M

Abstract
Botrytis cinerea causes pre- and postharvest decay of many fruit and vegetable crops. A survey in German strawberry fields revealed Botrytis strains that differed from B. cinerea in diagnostic PCR markers and growth appearance. Phylogenetic analyses showed these strains to belong to an undescribed species in Botrytis clade 2, named Botrytis fragariae sp. nov. Isolates of B. fragariae were detected in strawberry fields throughout Germany, sometimes at similar frequencies as B. cinerea, and in the Southeastern United States. B. fragariae was isolated from overwintering strawberry tissue, but not from freshly infected fruit. B. fragariae invaded strawberry tissues with similar or lower efficiency than B. cinerea but showed poor colonization of inoculated non-host plant tissues. These data and its exclusive occurrence on strawberry indicate that B. fragariae is host-specific and has a different tissue preference than B. cinerea Various fungicide resistance patterns were observed in B. fragariae populations. Many B. fragariae strains showed resistance to one or several chemical classes of fungicides, and an efflux-based multidrug resistance (MDR1) phenotype previously described for B. cinerea Resistance-related mutations in B. fragariae were identical or similar to those of B. cinerea for carbendazim (E198A mutation in tubA), azoxystrobin (G143A in cytB), iprodione (G367A+V368F in bos1) and MDR1 (gain-of-function mutations in the transcription factor mrr1 gene, and overexpression of the drug efflux transporter gene atrB). The widespread occurrence of B. fragariae indicates that this species is adapted to fungicide-treated strawberry fields and may be of local importance as a gray mold pathogen alongside B. cinereaImportance Gray mold is the most important fruit rot on strawberries worldwide, and requires fungicide treatments for control. For a long time it was believed to be caused only by Botrytis cinerea, a ubiquitous pathogen with broad host range which quickly develops fungicide resistance. We report the discovery and description of a new species, named Botrytis fragariae, which is widely distributed in commercial strawberry fields in Germany and the Southeastern United States. It was observed on overwintering tissue but not on freshly infected fruit and seems host-specific based on its occurrence and on artificial infection tests. B. fragariae has also developed resistance to several fungicides which is caused by similar mutations as those known for B. cinerea, including an efflux-based multidrug resistance. Our data indicate that B. fragariae could be of practical importance as a strawberry pathogen in some regions where it is similarly abundant as B. cinerea.

PMID: 28235878 [PubMed - as supplied by publisher]




Spread from the Sink to the Patient: in situ Study Using Green Fluorescent Protein (GFP) Expressing- Escherichia coli to Model Bacterial Dispersion from Hand Washing Sink Trap Reservoirs.
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Spread from the Sink to the Patient: in situ Study Using Green Fluorescent Protein (GFP) Expressing- Escherichia coli to Model Bacterial Dispersion from Hand Washing Sink Trap Reservoirs.

Appl Environ Microbiol. 2017 Feb 24;:

Authors: Kotay S, Chai W, Guilford W, Barry K, Mathers AJ

Abstract
There have been an increasing number of reports implicating Gammaproteobacteria often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand washing sink lab gallery to model dispersion of green fluorescent protein (GFP)- expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-E.coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-E.coli were allowed to mature in the P-trap under conditions similar to a hospital environment a GFP-E.coli containing putative biofilm extended upward over seven days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas (<30 inches) during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap resulting in droplet dispersion rather than directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient.Importance Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug resistant bacteria, which then result in hospital acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic resistant bacteria which can thrive in wastewater environments and cause infections in vulnerable patients.

PMID: 28235877 [PubMed - as supplied by publisher]




Comparison of Biochemical Properties of the Original and Newly Identified Oleate Hydratases from Stenotrophomonas maltophilia.
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Comparison of Biochemical Properties of the Original and Newly Identified Oleate Hydratases from Stenotrophomonas maltophilia.

Appl Environ Microbiol. 2017 Feb 24;:

Authors: Kang WR, Seo MJ, Shin KC, Park JB, Oh DK

Abstract
Oleate hydratases (OhyAs) catalyze the conversion of unsaturated fatty acids to 10-hydroxy fatty acids, which are used as precursors of important industrial compounds, including lactones, and ω-hydroxycarboxylic and α,ω-dicarboxylic acids. The genes encoding OhyA and a putative fatty acid hydratase in Stenotrophomonas maltophilia were identified by genomic analysis. The putative fatty acid hydratase was purified and identified as an oleate hydratase (OhyA2) based on its substrate specificity. The activity of OhyA2 as a holoenzyme was not affected on adding cofactors, whereas the activity of the original oleate hydratase (OhyA1) showed an increase. Thus, all characterized OhyAs were categorized as OhyA1 or OhyA2 based on the activities of holoenzymes on adding cofactors, which were determined by the type of the fourth conserved amino acid of FAD-binding motif. The hydration activity of S. maltophilia OhyA2 towards unsaturated fatty acids, including oleic acid, palmitoleic acid, linoleic acid, α-linolenic acid, and γ-linolenic acid, was greater than that of OhyA1. Moreover, the specific activity of S. maltophilia OhyA2 towards unsaturated fatty acids, with the exception of γ-linolenic acid, was the highest among all reported OhyAs.IMPORTANCE All characterized OhyAs were categorized as OhyA1s or OhyA2s based on the different properties of the reported and newly identified holo-OhyAs in S. maltophilia on addition of cofactors. OhyA2s showed higher activity towards polyunsaturated fatty acids (PUFAs), including linoleic acid, α-linolenic acid, and γ-linolenic acid, compared with those of OhyA1s. This suggests that OhyA2s can be used more effectively to convert plant oils to 10-hydroxy fatty acids because plant oils contain not only oleic acid but also PUFAs. The hydration activity of the newly identified OhyA2 from S. maltophilia towards oleic acid was the highest among the activities reported so far. Therefore, this enzyme is an efficient biocatalyst for the conversion of plant oils to 10-hydroxy fatty acids, which can be further converted to important industrial materials.

PMID: 28235876 [PubMed - as supplied by publisher]




Engineering Cyanobacterial Cell Morphology for Enhanced Recovery and Processing of Biomass.
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Engineering Cyanobacterial Cell Morphology for Enhanced Recovery and Processing of Biomass.

Appl Environ Microbiol. 2017 Feb 24;:

Authors: Jordan A, Chandler J, MacCready JS, Huang J, Osteryoung KW, Ducat DC

Abstract
Cyanobacteria are emerging as alternative crop species for the production of fuels, chemicals, and biomass. Yet, the success of these microbes depends upon the development of cost-effective technologies that permit scaled cultivation and cell harvesting. Here, we investigate the feasibility of engineering cell morphology in order to improve biomass recovery and decrease energetic costs associated with lysing cyanobacterial cells. Specifically, we modify the levels of Min system proteins in Synechococcus elongatus sp. PCC 7942. The Min system has established functions in controlling cell division by regulating assembly of FtsZ, a tubulin-like protein required to define the bacterial division plane. We show that altering expression of two FtsZ-regulatory proteins, MinC and Cdv3, permits control over cell morphology by disrupting FtsZ localization and cell division, without preventing continued cell growth. By varying the expression of these proteins, we can tune the length of cyanobacterial cells across a broad dynamic range: anywhere from a ∼20% increased length relative to wildtype to near-millimeter lengths. Highly elongated cells exhibit increased rates of sedimentation under low centrifugal forces or by gravity-assisted settling. Furthermore, hyperelongated cells are also more susceptible to lysis through the application of mild physical stress. Collectively, these results demonstrate a novel approach towards decreasing harvesting and processing costs associated with mass cyanobacterial cultivation through altering morphology at the cellular level.Importance: We show that the cell length of a model cyanobacterial species can be programmed through the rational manipulation of expression of protein factors that suppress cell division. In some instances, we are able to increase the size of these cells to near millimeter lengths through this approach. The resulting elongated cells have favorable properties with regard to cell harvesting and lysis. Furthermore cells treated in this manner continue to grow rapidly at time scales similar to those of uninduced controls. To our knowledge, this is the first reported example of engineering the cell morphology of cyanobacteria or algae to make them more compatible with downstream processing steps that present economic barriers to their use as alternative crop species. Therefore, our results are a promising proof-of-principle for the use of morphology engineering to increase the cost-effectiveness of mass cultivation of cyanobacteria for various sustainability initiatives.

PMID: 28235875 [PubMed - as supplied by publisher]




A Novel Subfamily Esterase with a Homoserine Transacetylase-like Fold but no Transferase Activity.
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A Novel Subfamily Esterase with a Homoserine Transacetylase-like Fold but no Transferase Activity.

Appl Environ Microbiol. 2017 Feb 24;:

Authors: Li PY, Yao QQ, Wang P, Zhang Y, Li Y, Zhang YQ, Hao J, Zhou BC, Chen XL, Shi M, Zhang YZ, Zhang XY

Abstract
Microbial esterases play important roles in deep-sea organic carbon degradation and cycling. Though sharing similar catalytic triads and oxyanion holes, esterases are hydrolases, and homoserine transacetylases (HTAs) are transferases. Because two HTA homologs were identified as acetyl esterases, the HTA family was divided into the bonafide acetyltransferase subfamily and the acetyl esterase subfamily recently. Here, we identified and characterized a novel HTA-like esterase, Est22, from a deep-sea sedimentary metagenomic library. Est22 could efficiently hydrolyze esters with acyl length of up to six carbon atoms, but had no transacetylase activity, which is different from HTAs and HTA-like acetyl esterases. Phylogenetic analysis also showed that Est22 and its homologs form a separate branch in the HTA family. We solved the structures of Est22 and its mutant L374D and modeled the structure of L374D with p-nitrophenyl butyrate. Based on structural, mutational and biochemical analyses, Phe(71) and Met(176) in the oxyanion hole and Arg(294) are revealed to be the key substrate-binding residues. A detailed structural comparison indicated that differences in their catalytic tunnels lead to the different substrate specificities of Est22 and the other two HTA subfamilies. Biochemical and sequence analyses suggested that Est22 homologs may share the same substrate recognition and catalysis mechanism with Est22. Due to the significant differences in sequence, structure and substrate specificity between Est22 (and its homologs) and the other two HTA subfamilies, we suggest that Est22 and its homologs represent a new subfamily in the HTA family.IMPORTANCE Microbial esterases play important roles in the turnover of organic carbon in deep sea. Esterases and homoserine transacetylases (HTAs) represent two groups of α/β hydrolases. Esterases catalyze the hydrolysis of simple esters and are widely used in pharmaceutical and agrochemical industries, while HTAs catalyze the transfer of acetyl group from acetyl-CoA to homoserine and are essential for microbial growth. Here, we reported a novel HTA-like esterase, Est22, from a deep-sea sediment. Because of the significant differences in sequence, structure and substrate specificity from HTAs and HTA-like acetyl esterases, Est22 together with its homologs represent a new subfamily in the HTA family. This study offers new knowledge on marine esterases.

PMID: 28235874 [PubMed - as supplied by publisher]




High resolution X-ray structures of two functionally distinct members of the cyclic amide hydrolase (CyAH) family of Toblerone fold enzymes.
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High resolution X-ray structures of two functionally distinct members of the cyclic amide hydrolase (CyAH) family of Toblerone fold enzymes.

Appl Environ Microbiol. 2017 Feb 24;:

Authors: Peat TS, Balotra S, Wilding M, Hartley CJ, Newman J, Scott C

Abstract
The Toblerone-fold was discovered recently when the first structure of the cyclic amide hydrolase, AtzD (a cyanuric acid hydrolase), was elucidated. We surveyed the cyclic amide hydrolase family finding a strong correlation between phylogenetic distribution and specificity for either cyanuric acid or barbituric acid; albeit, one of six classes (IV) could not be tested due to lack of expression of the proteins from it, and another class (V) had neither cyanuric acid nor barbituric acid hydrolase activity. High resolution X-ray structures were obtained for a class VI barbituric acid hydrolase (1.7 Å) from a Rhodococcus sp. and a class V cyclic amide hydrolase (2.4 Å) from a Frankia sp. for which we were unable to identify a substrate. Both structures were homologous with the tetrameric Toblerone-fold enzyme AtzD, demonstrating a high degree of structural conservation within the cyclic amide hydrolase family. The barbituric acid hydrolase structure did not contain zinc, in contrast with early reports of zinc-dependent activity for this enzyme. Instead, each barbituric acid hydrolase monomer contained either a Na(+) or a Mg(2+), analogous to the structural metal found in cyanuric acid hydrolase. The Frankia cyclic amide hydrolase contained no metal, but instead formed unusual, reversible, intermolecular vicinal disulfide bonds that contributed to the thermal stability of the protein. The active sites were largely conserved between the three enzymes, differing at six positions, which likely determine substrate specificity.Importance The 'Toblerone-fold' enzymes catalyze an unusual ring-opening hydrolysis with cyclic amide substrates. A survey of these enzymes shows that there is a good correlation between physiological function and phylogenetic distribution within this family of enzymes, and provide insights into the evolutionary relationships between the cyanuric acid and barbituric acid hydrolases.This family of enzymes is structurally and mechanistically distinct from other enzyme families; however, to date the structure of just two, physiologically identical, enzymes from this family have been described. We present two new structures: a barbituric acid hydrolase and an enzyme of unknown function. These structures confirm that the CyAH family share the unusual Toblerone fold, albeit with some significant differences.

PMID: 28235873 [PubMed - as supplied by publisher]




Role and characterization of a cyanide induced 3-cyanoalanine nitrilase in the cyanide assimilating bacterium Pseudomonas pseudoalcaligenes CECT 5344.
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Role and characterization of a cyanide induced 3-cyanoalanine nitrilase in the cyanide assimilating bacterium Pseudomonas pseudoalcaligenes CECT 5344.

Appl Environ Microbiol. 2017 Feb 24;:

Authors: Acera F, Carmona MI, Castillo F, Quesada A, Blasco R

Abstract
Pseudomonas pseudoalcaligenes CECT 5344 is a bacterium able to assimilate cyanide as the sole nitrogen source. Under this growth conditions, a 3-cyanoalanine nitrilase enzymatic activity was induced. This activity was coded by nit4, one of the four nitrilases detected in the genome of this bacterium, and its expression in E. coli enabled the recombinant strain to fully assimilate 3-cyanoalanine. P. pseudoalcaligenes CECT 5344 showed a weak growth level with 3-cyanoalanine as N-source, unless KCN was also added. Moreover, a knockout mutant of P. pseudoalcaligenes CECT5344 in the nit4 gene became severely impaired to grow with 3-cyanoalanine and cyanide as nitrogen sources. The native enzyme expressed in E. coli was purified up to electrophoretic homogeneity and biochemically characterized. Nit4 seems to be specific for 3-cyanoalanine and the amount of ammonium derived from the enzymatic activity doubled in the presence of exogenously added asparaginase activity, which demonstrated that Nit4 enzyme had both 3-cyanoalanine nitrilase and hydratase activity. nit4 gene is located downstream the cyanide-resistance transcriptional unit encoding cio1 genes, whose expression is under the positive control of cyanide. Real time PCR experiments revealed that nit4 expression was also positively regulated by cyanide, both in minimal and LB medium. These results suggest that this gene cluster including cio1 and nit4 could be involved both in cyanide resistance and its assimilation by P. pseudoalcaligenes CECT 5344.IMPORTANCE Cyanide is a highly toxic molecule present in some industrial wastes due to its application in several manufacturing processes, such as gold-mining and electroplating industry. The biodegradation of cyanide from contaminated wastes could be an attractive alternative to their physicochemical treatments. P. pseudoalcaligenes CECT 5344 is a bacterial strain able to assimilate cyanide under alkaline conditions, thus avoiding its volatilization as HCN. This manuscript describes and characterizes an enzyme (Nit4), induced by cyanide, that is probably involved on cyanide assimilation. The biochemical characterization of Nit4 provides a segment for building a cyanide assimilation pathway in P. pseudoalcaligenes This information could be useful for understanding, and hopefully improving, the mechanisms involved in bacterial cyanide biodegradation and its application in the treatment of cyanide-containing wastes.

PMID: 28235872 [PubMed - as supplied by publisher]




Metatranscriptomic profiling reveals linkages between the active rumen microbiome and feed efficiency in beef cattle.
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Metatranscriptomic profiling reveals linkages between the active rumen microbiome and feed efficiency in beef cattle.

Appl Environ Microbiol. 2017 Feb 24;:

Authors: Li F, Guan LL

Abstract
Exploring compositional and functional characteristics of the rumen microbiome can improve the understanding of its role in rumen function and feed efficiency. In this study, we applied metatranscriptomics to characterize the active rumen microbiomes of beef cattle with different feed efficiencies (efficient, n=10; inefficient, n=10) using total RNA sequencing. Active bacterial and archaeal compositions were estimated based on 16S rRNAs, and active microbial metabolic functions including carbohydrate-active enzymes (CAZymes) were assessed based on mRNAs from the same metatranscriptomic datasets. In total, six bacterial phyla (Proteobacteria, Firmicutes, Bacteroidetes, Spirochaetes, Cyanobacteria, and Synergistetes), eight bacterial families (Succinivibrionaceae, Prevotellaceae, Ruminococcaceae, Lachnospiraceae, Veillonellaceae, Spirochaetaceae, Dethiosulfovibrionaceae, and Mogibacteriaceae), four archaeal clades (Methanomassiliicoccales, Methanobrevibacter ruminantium, Methanobrevibacter gottschalki, and Methanosphaera), 112 metabolic pathways, and 126 CAZymes were identified as core components of the active rumen microbiome. Through comparative analysis, three bacterial families (Lachnospiraceae, Lactobacillaceae, and Veillonellaceae) tended to be more abundant in low-feed efficiency animals (P < 0.10) and one archaeal taxa (Methanomassiliicoccale) tended to more abundant in efficient cattle (P < 0.10). Meanwhile, 32 microbial metabolic pathways and 12 CAZymes were differentially abundant (linear discriminant analysis score > 2 with P < 0.05) between two groups. Among them, 30 metabolic pathways and 11 CAZymes were more abundant in the rumen of inefficient cattle, while 2 metabolic pathways and 1 CAZymes were more abundant in efficient animals. These findings suggest that the rumen microbiomes of inefficient cattle may have more diverse activities than those of efficient cattle, which may be related to the host feed efficiency variation.Importance This study applied total RNA-based metatranscriptomics and showed the linkage between the active rumen microbiome with feed efficiency (residual feed intake) in beef cattle. The data generated from current study provide fundamental information on active rumen microbiome at both compositional and functional levels, which serve as a foundation to study rumen function and its role in feed efficiency. The findings on the active rumen microbiome that may contribute to variations in feed efficiency of beef cattle highlight the possibility to enhance nutrient utilization and improve cattle feed efficiency through altering rumen microbial functions.

PMID: 28235871 [PubMed - as supplied by publisher]