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Objectives: Morphologic anomalies of the feline gallbladder (GB) have been previously reported in the literature. These morphologic variants are frequently encountered on routine abdominal ultrasound examination. The aim of this study was to provide an ultrasonographic classification system of these variants and document the overall incidence in the feline population. Methods: A prospective, descriptive study was undertaken; cats that had an abdominal ultrasound examination that included at least one sagittal and transverse plane image of the GB were included. GB shape was evaluated and categorized based on a classification scheme of morphologic variants modified from the human literature. Septated (S), bilobed (B1, B2, B3), duplex (D) and complex (C) categories were described. Results: Of 516 cats included in the study, 389 had normal GB morphology, while 127 had anomalous GB morphology. The overall incidence rate of anomalous GB morphology was 24.61%. When examined by morphologic type, the septated (S) morphology had an incidence of 9.69%. A bilobed (B) morphology was the most commonly observed classification; incidence was 14.35% within our population; incidence of B1, B2 and B3 subtypes were 2.91%, 6.98% and 4.46%, respectively. Duplex GBs only made up 0.39% of the total population. The incidence of complex (C) morphologies was 0.19%. Conclusions and relevance: The incidence of GB morphologic anomalies was higher in our population than previously reported. Identification of these anomalies on routine ultrasound evaluation is common; numerous different morphologies can be identified and a standardized classification scheme is proposed. Complete evaluation of morphology can be challenging, particularly with regard to cystic duct anatomy. Clinical significance is uncertain and future studies are warranted to determine the relationship between morphologic variants and hepatobiliary disease. [ABSTRACT FROM AUTHOR]

In many organisms, developmentally programmed double-strand breaks (DSBs) formed by the SPO11 transesterase initiate meiotic recombination, which promotes pairing and segregation of homologous chromosomes (1). Because every chromosome must receive a minimum number of DSBs, attention has focused on factors that support DSB formation (2). However, improperly repaired DSBs can cause meiotic arrest or mutation (3,4); thus, having too many DSBs is probably as deleterious as having too few. Only a small fraction of SPO11 protein ever makes a DSB in yeast or mouse (5) and SPO11 and its accessory factors remain abundant long after most DSB formation ceases (1), implying the existence of mechanisms that restrain SPO11 activity to limit DSB numbers. Here we report that the number of meiotic DSBs in mouse is controlled by ATM, a kinase activated by DNA damage to trigger checkpoint signalling and promote DSB repair. Levels of SPO11-oligonucleotide complexes, by-products of meiotic DSB formation, are elevated at least tenfold in spermatocytes lacking ATM. Moreover, Atm mutation renders SPO11-oligonucleotide levels sensitive to genetic manipulations that modulate SPO11 protein levels. We propose that ATM restrains SPO11 via a negative feedback loop in which kinase activation by DSBs suppresses further DSB formation. Our findings explain previously puzzling phenotypes of Atm-null mice and provide a molecular basis for the gonadal dysgenesis observed in ataxia telangiectasia, the human syndrome caused by ATM deficiency., SPO11 creates DSBs via a covalent protein-DNA intermediate that is endonucleolytically cleaved to release SPO11 attached to a short oligonucleotide, freeing DSB ends for further processing and recombination (5) (Fig. [...]

A study was conducted to examine the chemical composition of extracellular polymeric substances (EPS) extracted from two natural microbial pellicle biofilms growing on acid mine drainage (AMD) solutions. The results indicated that the biochemical composition of the EPS from the acidic biofilms is dependent on maturity and is regulated by the microbial communities as well as the local geochemical environment.

The analysis of near-complete population (composite) genomic sequences for coexisting acidophilic iron-oxidizing Leptospirillum group II and III bacteria (phylum Nitrospirae) and an extrachromosomal plasmid from a Richmnd Mine, Iron Mountain, Ca, acid mine drainage biofilm is presented. Both the bacterial groups are found to have genes for community-essential functions, including carbon fixation and biosynthesis of vitamins, fatty acids, and biopolymers and multiple pathways for osmotic protection.

The characterization of a novel, soluble cytochrome with an unusual visible signature at 579 nm (Cyt(579)) is described. The results suggested that Cyt(579) plays an important role as an electron transfer protein to shuttle electrons derived from Fe(II) oxidation to support critical metabolical functions in the acidophilic microbial community.

Background: New lignocellulolytic enzymes are needed that maintain optimal activity under the harsh conditions present during industrial enzymatic deconstruction of biomass, including high temperatures, the absence of free water, and the presence of inhibitors from the biomass. Enriching lignocellulolytic microbial communities under these conditions provides a source of microorganisms that may yield robust lignocellulolytic enzymes tolerant to the extreme conditions needed to improve the throughput and efficiency of biomass enzymatic deconstruction. Identification of promising enzymes from these systems is challenging due to complex substrate-enzyme interactions and requirements to assay for activity. In this study, metatranscriptomes from compost-derived microbial communities enriched on rice straw under thermophilic and mesophilic conditions were sequenced and analyzed to identify lignocellulolytic enzymes overexpressed under thermophilic conditions. To determine differential gene expression across mesophilic and thermophilic treatments, a method was developed which pooled gene expression by functional category, as indicated by Pfam annotations, since microbial communities performing similar tasks are likely to have overlapping functions even if they share no specific genes. Results: Differential expression analysis identified enzymes from glycoside hydrolase family 48, carbohydrate binding module family 2, and carbohydrate binding module family 33 domains as significantly overexpressed in the thermophilic community. Overexpression of these protein families in the thermophilic community resulted from expression of a small number of genes not currently represented in any protein database. Genes in overexpressed protein families were predominantly expressed by a single Actinobacteria genus, Micromonospora. Conclusions: Coupling measurements of deconstructive activity with comparative analyses to identify overexpressed enzymes in lignocellulolytic communities provides a targeted approach for discovery of candidate enzymes for more efficient biomass deconstruction. Glycoside hydrolase family 48 cellulases and carbohydrate binding module family 33 polysaccharide monooxygenases with carbohydrate binding module family 2 domains may improve saccharification of lignocellulosic biomass under high-temperature and low moisture conditions relevant to industrial biofuel production. [ABSTRACT FROM AUTHOR]

The ability to solubilize lignocellulose makes certain ionic liquids (ILs) very effective reagents for pretreating biomass prior to its saccharification for biofuel fermentation. However, residual IL in the aqueous sugar solution can inhibit the growth and function of biofuel-producing microorganisms. In E. coli this toxicity can be partially overcome by the heterologous expression of an IL efflux pump encoded by eilA from Enterobacter lignolyticus. In the present work, we used microarray analysis to identify native E. coli IL-inducible promoters and develop control systems for regulating eilA gene expression. Three candidate promoters, PmarR’, PydfO’, and PydfA’, were selected and compared to the IPTG-inducible PlacUV5 system for controlling expression of eilA. The PydfA’ and PmarR’ based systems are as effective as PlacUV5 in their ability to rescue E. coli from typically toxic levels of IL, thereby eliminating the need to use an IPTG-based system for such tolerance engineering. We present a mechanistic model indicating that inducible control systems reduce target gene expression when IL levels are low. Selected-reaction monitoring mass spectrometry analysis revealed that at high IL concentrations EilA protein levels were significantly elevated under the control of PydfA’ and PmarR’ in comparison to the other promoters. Further, in a pooled culture competition designed to determine fitness, the strain containing pPmarR’-eilA outcompeted strains with other promoter constructs, most significantly at IL concentrations above 150 mM. These results indicate that native promoters such as PmarR’ can provide effective systems for regulating the expression of heterologous genes in host engineering and simplify the development of industrially useful strains. [ABSTRACT FROM AUTHOR]

High-solids incubations were performed to enrich for microbial communities and enzymes that decompose rice straw under mesophilic (35°C) and thermophilic (55°C) conditions. Thermophilic enrichments yielded a community that was 7.5 times more metabolically active on rice straw than mesophilic enrichments. Extracted xylanase and endoglucanse activities were also 2.6 and 13.4 times greater, respectively, for thermophilic enrichments. Metagenome sequencing was performed on enriched communities to determine community composition and mine for genes encoding lignocellulolytic enzymes. Proteobacteria were found to dominate the mesophilic community while Actinobacteria were most abundant in the thermophilic community. Analysis of protein family representation in each metagenome indicated that cellobiohydrolases containing carbohydrate binding module 2 (CBM2) were significantly overrepresented in the thermophilic community. Micromonospora, a member of Actinobacteria, primarily housed these genes in the thermophilic community. In light of these findings, Micromonospora and other closely related Actinobacteria genera appear to be promising sources of thermophilic lignocellulolytic enzymes for rice straw deconstruction under high-solids conditions. Furthermore, these discoveries warrant future research to determine if exoglucanases with CBM2 represent thermostable enzymes tolerant to the process conditions expected to be encountered during industrial biofuel production. [ABSTRACT FROM AUTHOR]

Enzyme catalyzed conversion of plant biomass to sugars is an inherently inefficient process, and one of the major factors limiting economical biofuel production. This is due to the physical barrier presented by polymers in plant cell walls, including semi-crystalline cellulose, to soluble enzyme accessibility. In contrast to the enzymes currently used in industry, bacterial cellulosomes organize cellulases and other proteins in a scaffold structure, and are highly efficient in degrading cellulose. To mimic this clustered assembly of enzymes, we conjugated cellulase obtained from Trichoderma viride to polystyrene nanospheres (cellulase:NS) and tested the hydrolytic activity of this complex on cellulose substrates from purified and natural sources. Cellulase:NS and free cellulase were equally active on soluble carboxymethyl cellulose (CMC); however, the complexed enzyme displayed a higher affinity in its action on microcrystalline cellulose. Similarly, we found that the cellulase:NS complex was more efficient in degrading natural cellulose structures in the thickened walls of cultured wood cells. These results suggest that nanoparticle-bound enzymes can improve catalytic efficiency on physically intractable substrates. We discuss the potential for further enhancement of cellulose degradation by physically clustering combinations of different glycosyl hydrolase enzymes, and applications for using cellulase:NS complexes in biofuel production. [ABSTRACT FROM AUTHOR]

During microbial evolution, genome rearrangement increases with increasing sequence divergence. If the relationship between synteny and sequence divergence can be modeled, gene clusters in genomes of distantly related organisms exhibiting anomalous synteny can be identified and used to infer functional conservation. We applied the phylogenetic pairwise comparison method to establish and model a strong correlation between synteny and sequence divergence in all 634 available Archaeal and Bacterial genomes from the NCBI database and four newly assembled genomes of uncultivated Archaea from an acid mine drainage (AMD) community. In parallel, we established and modeled the trend between synteny and functional relatedness in the 118 genomes available in the STRING database. By combining these models, we developed a gene functional annotation method that weights evolutionary distance to estimate the probability of functional associations of syntenous proteins between genome pairs. The method was applied to the hypothetical proteins and poorly annotated genes in newly assembled acid mine drainage Archaeal genomes to add or improve gene annotations. This is the first method to assign possible functions to poorly annotated genes through quantification of the probability of gene functional relationships based on synteny at a significant evolutionary distance, and has the potential for broad application. INSET: Author Summary. [ABSTRACT FROM AUTHOR]

Characterizing proteins recovered from natural microbial communities affords the opportunity to correlate protein expression and modification with environmental factors, including species composition and successional stage. Proteogenomic and biochemical studies of pellicle biofilms from subsurface acid mine drainage streams have shown abundant cytochromes from the dominant organism, Leptospirillum Group II. These cytochromes are proposed to be key proteins in aerobic Fe(II) oxidation, the dominant mode of cellular energy generation by the biofilms. In this study, we determined that posttranslational modification and expression of amino-acid sequence variants change as a function of biofilm maturation. For Cytochrome579 (Cyt579), the most abundant cytochrome in the biofilms, late developmental-stage biofilms differed from early-stage biofilms in N-terminal truncations and decreased redox potentials. Expression of sequence variants of two monoheme c-type cytochromes also depended on biofilm development. For Cyt572, an abundant membrane-bound cytochrome, the expression of multiple sequence variants was observed in both early and late developmental-stage biofilms; however, redox potentials of Cyt572 from these different sources did not vary significantly. These cytochrome analyses show a complex response of the Leptospirillum Group II electron transport chain to growth within a microbial community and illustrate the power of multiple proteomics techniques to define biochemistry in natural systems. [ABSTRACT FROM AUTHOR]

Pellicle biofilms colonize the air–solution interface of underground acid mine drainage (AMD) streams and pools within the Richmond Mine (Iron Mountain, Redding, CA, USA). They exhibit relatively low species richness and, consequently, represent good model systems to study natural microbial community structure. Fluorescence in situ hybridization combined with epifluorescent microscopy and transmission electron microscopy revealed spatially and temporally defined microbial assemblages. Leptospirillum group II dominates the earliest developmental stages of stream pellicles. With increasing biofilm maturity, the proportion of archaea increases in conjunction with the appearance of eukaryotes. In contrast, mature pool pellicles are stratified with a densely packed bottom layer of Leptospirillum group II, a less dense top layer composed mainly of archaea and no eukarya. Immunohistochemical detection of Leptospirillum group II cytochrome 579 indicates a high abundance of this protein at the interface of the biofilm with the AMD solution. Consequently, community architecture, which most likely develops in response to chemical gradients across the biofilm, is reflected at the functional gene expression level.The ISME Journal (2009) 3, 266–270; doi:10.1038/ismej.2008.90; published online 9 October 2008 [ABSTRACT FROM AUTHOR]

Deep sequencing of a low-complexity microbial community revealed extensive recombination as well as polymorphic and gene content variation between individuals of the dominant organism. We show that strains defined by linked polymorphisms are not maintained by positive selection; instead, they are predominantly maintained by the forces of migration and drift. [ABSTRACT FROM AUTHOR]

Recently, there has been intense interest in the role of electron transfer by microbial communities in biogeochemical systems. We examined the process of iron oxidation by microbial biofilms in one of the most extreme environments on earth, where the inhabited water is pH 0.5–1.2 and laden with toxic metals. To approach the mechanism of Fe(II) oxidation as a means of cellular energy acquisition, we isolated proteins from natural samples and found a conspicuous and novel cytochrome, Cyt572, which is unlike any known cytochrome. Both the character of its covalently bound prosthetic heme group and protein sequence are unusual. Extraction of proteins directly from environmental biofilm samples followed by membrane fractionation, detergent solubilization and gel filtration chromatography resulted in the purification of an abundant yellow-red protein. The purified protein has a cytochrome c-type heme binding motif, CxxCH, but a unique spectral signature at 572 nm, and thus is called Cyt572. It readily oxidizes Fe2+ in the physiologically relevant acidic regime, from pH 0.95–3.4. Other physical characteristics are indicative of a membrane-bound multimeric protein. Circular dichroism spectroscopy indicates that the protein is largely beta-stranded, and 2D Blue-Native polyacrylamide gel electrophoresis and chemical crosslinking independently point to a multi-subunit structure for Cyt572. By analyzing environmental genomic information from biofilms in several distinctly different mine locations, we found multiple genetic variants of Cyt572. MS proteomics of extracts from these biofilms substantiated the prevalence of these variants in the ecosystem. Due to its abundance, cellular location and Fe2+ oxidation activity at very low pH, we propose that Cyt572 provides a critical function for fitness within the ecological niche of these acidophilic microbial communities.The ISME Journal (2008) 2, 542–550; doi:10.1038/ismej.2008.17; published online 28 February 2008 [ABSTRACT FROM AUTHOR]

Replication and related processes in eukaryotic cells require replication factor C (RFC) to load a molecular clamp for DNA polymerase in an ATP-driven process, involving multiple molecular interactions. The detailed understanding of this mechanism is hindered by the lack of data regarding structure, mutual arrangement, and dynamics of the players involved. In this study, we analyzed interactions that take place during loading onto DNA of either the PCNA clamp or the Rad9-Rad1-Hus1 checkpoint complex, using computationally derived molecular models. Combining the modeled structures for each RFC subunit with known structural, biochemical, and genetic data, we propose detailed models of how two of the RFC subunits, RFC1 and RFC3, interact with the C-terminal regions of PCNA. RFC1 is predicted to bind PCNA similarly to the p21-PCNA interaction, while the RFC3-PCNA binding is proposed to be similar to the E. coli δ-β interaction. Additional sequence and structure analysis, supported by experimental data, suggests that RFC5 might be the third clamp loader subunit to bind the equivalent PCNA region. We discuss functional implications stemming from the proposed model of the RFC1-PCNA interaction and compare putative clamp-interacting regions in RFC1 and its paralogs, Rad17 and Ctf18. Based on the individual intermolecular interactions, we propose RFC and PCNA arrangement that places three RFC subunits in association with each of the three C-terminal regions in PCNA. The two other RFC subunits are positioned at the two PCNA interfaces, with the third PCNA interface left unobstructed. In addition, we map interactions at the level of individual subunits between the alternative clamp loader/clamp system, Rad17-RFC2-5/Rad9-Rad1-Hus1. The proposed models of interaction between two clamp/clamp loader pairs provide both structural framework for interpretation of existing experimental data and a number of specific findings that can be subjected to direct experimental testing. [ABSTRACT FROM AUTHOR]

Microalgae have major functions in global biogeochemical cycles and are promising sources of renewable energy, yet the relationships between algal hosts and their associated microbiomes remain relatively underexplored. Understanding community organization of microalgal microbiomes, such as how algal species identity influences bacterial community structure, will aid in efforts to engineer more efficient phototrophic ecosystems. Here, we examined the community assembly of phycosphere-associated (attached) and free-living bacterial taxa associated with two marine microalgae: the diatom Phaeodactylum tricornutum and eustigmatophyte Microchloropsis salina. Samples were collected from outdoor mesocosms, raceway ponds, and laboratory enrichments, and bacterial taxa identified by 16S rRNA gene sequences. In outdoor mesocosms, we found distinct bacterial taxa associated with each algal species, including the Cytophagaceae and Rhodobacteraceae families with P. tricornutum , and Rhodobacteraceae, Hyphomonadaceae, and Saprospiraceae with M. salina. Additionally, there were host-specific differences in the bacterial genera associated with the phycosphere, including Novosphingobium and Rhodopirellula with P. tricornutum , and Methylophaga and Dyadobacter with M. salina. Bacterial communities from outdoor monoalgal P. tricornutum and polyalgal P. tricornutum / M. salina samples were used as inocula for laboratory enrichments with axenic P. tricornutum. Here, similar bacterial communities emerged, suggesting that the algal host exerts substantial influence over bacterial community assembly. Further enrichments for phycosphere-association revealed differing outcomes of community assembly processes contingent on the initial community composition. Phycosphere-associated communities from monoalgal P. tricornutum mesocosms were highly similar to one another, suggesting deterministic processes, whereas cultures from mixed M. salina / P. tricornutum raceways followed two apparent paths differentiated by the stochastic loss of specific community members and convergence towards or further deviation from the monoalgal samples. These results demonstrate that algal-associated bacterial communities are controlled by algal host, culture conditions, and the initial inoculum composition of the algal microbiome, and this knowledge can inform the engineering of more productive algal systems. • Microalgae influence bacterial community compositions. • Different microalgae select for different phycosphere-associated bacteria. • Stochastic events can lead to multiple stable community compositions. [ABSTRACT FROM AUTHOR]

The article presents a study focusing on global transcriptome response to ionic liquid by enterobacter lignolyticus bacterium. It shows that increasing ionic liquid 1-ethyl-3-methylimidazolium chloride concentrations caused a change in SCF1 cell membrane composition by increasing cyclopropane fatty acids that help in stabilizing the cell membrane. It concludes that the bacteria can resist the toxic effects of ionic liquid by changing membrane permeability.

Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from Zinnia elegans. Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulose-binding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis, but realistic enough to capture the natural complexity of lignocellulose in the plant cell wall. Consequently, these cells represent a suitable model for analyzing native lignocellulose degradation. [ABSTRACT FROM AUTHOR]

Abstract: Here we report a new microfluidic microbial fuel cell (MFC) platform built by soft-lithography techniques. The MFC design includes a unique sub-5μL polydimethylsiloxane soft chamber featuring carbon cloth electrodes and microfluidic delivery of electrolytes. Bioelectricity was generated using Shewanella oneidensis MR-1 cultivated on either complex organic substrates or lactate-based minimal medium. These micro-MFCs exhibited fast start-ups, reproducible current generation, and enhanced power densities up to 62.5Wm−3 that represents the best result for sub-100μL MFCs. Systematic comparisons of custom-made MFC reactors having different chamber sizes indicate volumetric power density is inversely correlated with chamber size in our systems: i.e., the smaller the chamber, the higher the power density is achieved. [Copyright &y& Elsevier]

Imidazolium ionic liquids (IILs) have a range of biotechnological applications, including as pretreatment solvents that extract cellulose from plant biomass for microbial fermentation into sustainable bioenergy. However, residual levels of IILs, such as 1-ethyl-3-methylimidazolium chloride ([C2C1im]Cl), are toxic to biofuel-producing microbes, including the yeast Saccharomyces cerevisiae. S. cerevisiae strains isolated from diverse ecological niches differ in genomic sequence and in phenotypes potentially beneficial for industrial applications, including tolerance to inhibitory compounds present in hydrolyzed plant feedstocks. We evaluated .100 genome-sequenced S. cerevisiae strains for tolerance to [C2C1im]Cl and identified one strain with exceptional tolerance. By screening a library of genomic DNA fragments from the [C2C1im]Cl-tolerant strain for improved IIL tolerance, we identified SGE1, which encodes a plasma membrane multidrug efflux pump, and a previously uncharacterized gene that we named ionic liquid tolerance 1 (ILT1), which encodes a predicted membrane protein. Analyses of SGE1 sequences from our panel of S. cerevisiae strains together with growth phenotypes implicated two single nucleotide polymorphisms (SNPs) that associated with IIL tolerance and sensitivity. We confirmed these phenotypic effects by transferring the SGE1 SNPs into a [C2C1im]Cl-sensitive yeast strain using CRISPR/Cas9 genome editing. Further studies indicated that these SNPs affect Sge1 protein stability and cell surface localization, influencing the amount of toxic IILs that cells can pump out of the cytoplasm. Our results highlight the general potential for discovering useful biotechnological functions from untapped natural sequence variation and provide functional insight into emergent SGE1 alleles with reduced capacities to protect against IIL toxicity. [ABSTRACT FROM AUTHOR]