Genomic and transcriptomic analysis of carbohydrate utilization by Paenibacillus sp. JDR-2: systems for bioprocessing plant polysaccharides.

BACKGROUND: Polysaccharides comprising plant biomass are potential resources for conversion to fuels and chemicals. These polysaccharides include xylans derived from the hemicellulose of hardwoods and grasses, soluble ß-glucans from cereals and starch as the primary form of energy storage in plants. Paenibacillus sp. JDR-2 (Pjdr2) has evolved a system for bioprocessing xylans. The central component of this xylan utilization system is a multimodular glycoside hydrolase family 10 (GH10) endoxylanase with carbohydrate binding modules (CBM) for binding xylans and surface layer homology (SLH) domains for cell surface anchoring. These attributes allow efficient utilization of xylans by generating oligosaccharides proximal to the cell surface for rapid assimilation. Coordinate expression of genes in response to growth on xylans has identified regulons contributing to depolymerization, importation of oligosaccharides and intracellular processing to generate xylose as well as arabinose and methylglucuronate. The genome of Pjdr2 encodes several other putative surface anchored multimodular enzymes including those for utilization of ß-1,3/1,4 mixed linkage soluble glucan and starch. RESULTS: To further define polysaccharide utilization systems in Pjdr2, its transcriptome has been determined by RNA sequencing following growth on barley-derived soluble ß-glucan, starch, cellobiose, maltose, glucose, xylose and arabinose. The putative function of genes encoding transcriptional regulators, ABC transporters, and glycoside hydrolases belonging to the corresponding substrate responsive regulon were deduced by their coordinate expression and locations in the genome. These results are compared to observations from the previously defined xylan utilization systems in Pjdr2. The findings from this study show that Pjdr2 efficiently utilizes these glucans in a manner similar to xylans. From transcriptomic and genomic analyses we infer a common strategy evolved by Pjdr2 for efficient bioprocessing of polysaccharides. CONCLUSIONS: The barley ß-glucan and starch utilization systems in Pjdr2 include extracellular glycoside hydrolases bearing CBM and SLH domains for depolymerization of these polysaccharides. Overlapping regulation observed during growth on these polysaccharides suggests they are preferentially utilized in the order of starch before xylan before barley ß-glucan. These systems defined in Pjdr2 may serve as a paradigm for developing biocatalysts for efficient bioprocessing of plant biomass to targeted biofuels and chemicals.

A 1,3-1,4-ß-Glucan Utilization Regulon in Paenibacillus sp. Strain JDR-2.

Paenibacillus sp. strain JDR-2 (Paenibacillus JDR-2) secretes a multimodular cell-associated glycoside hydrolase family 10 (GH10) endoxylanase (XynA10A1) that catalyzes the depolymerization of methylglucuronoxylan (MeGXn) and rapidly assimilates the products of depolymerization. Efficient utilization of MeGXn has been postulated to result from the coupling of the processes of exocellular depolymerization and assimilation of oligosaccharide products, followed by intracellular metabolism. Growth and substrate utilization patterns with barley glucan and laminarin similar to those observed with MeGXn as a substrate suggest similar processes for 1,3-1,4-ß-glucan and 1,3-ß-glucan depolymerization and product assimilation. The Paenibacillus JDR-2 genome includes a cluster of genes encoding a secreted multimodular GH16 ß-glucanase (Bgl16A1) containing surface layer homology (SLH) domains, a secreted GH16 ß-glucanase with only a catalytic domain (Bgl16A2), transporter proteins, and transcriptional regulators. Recombinant Bgl16A1 and Bgl16A2 catalyze the formation of trisaccharides, tetrasaccharides, and larger oligosaccharides from barley glucan and of mono-, di-, tri-, and tetrasaccharides and larger oligosaccharides from laminarin. The lack of accumulation of depolymerization products during growth and a marked preference for polymeric glucan over depolymerization products support a process coupling extracellular depolymerization, assimilation, and intracellular metabolism for ß-glucans similar to that ascribed to the GH10/GH67 xylan utilization system in Paenibacillus JDR-2. Coordinate expression of genes encoding GH16 ß-glucanases, transporters, and transcriptional regulators supports their role as a regulon for the utilization of soluble ß-glucans. As in the case of the xylan utilization regulons, this soluble ß-glucan regulon provides advantages in the growth rate and yields on polymeric substrates and may be exploited for the efficient conversion of plant-derived polysaccharides to targeted products.

Comparative genomics reveals diversity among xanthomonads infecting tomato and pepper.

BACKGROUND: Bacterial spot of tomato and pepper is caused by four Xanthomonas species and is a major plant disease in warm humid climates. The four species are distinct from each other based on physiological and molecular characteristics. The genome sequence of strain 85-10, a member of one of the species, Xanthomonas euvesicatoria (Xcv) has been previously reported. To determine the relationship of the four species at the genome level and to investigate the molecular basis of their virulence and differing host ranges, draft genomic sequences of members of the other three species were determined and compared to strain 85-10. RESULTS: We sequenced the genomes of X. vesicatoria (Xv) strain 1111 (ATCC 35937), X. perforans (Xp) strain 91-118 and X. gardneri (Xg) strain 101 (ATCC 19865). The genomes were compared with each other and with the previously sequenced Xcv strain 85-10. In addition, the molecular features were predicted that may be required for pathogenicity including the type III secretion apparatus, type III effectors, other secretion systems, quorum sensing systems, adhesins, extracellular polysaccharide, and lipopolysaccharide determinants. Several novel type III effectors from Xg strain 101 and Xv strain 1111 genomes were computationally identified and their translocation was validated using a reporter gene assay. A homolog to Ax21, the elicitor of XA21-mediated resistance in rice, and a functional Ax21 sulfation system were identified in Xcv. Genes encoding proteins with functions mediated by type II and type IV secretion systems have also been compared, including enzymes involved in cell wall deconstruction, as contributors to pathogenicity. CONCLUSIONS: Comparative genomic analyses revealed considerable diversity among bacterial spot pathogens, providing new insights into differences and similarities that may explain the diverse nature of these strains. Genes specific to pepper pathogens, such as the O-antigen of the lipopolysaccharide cluster, and genes unique to individual strains, such as novel type III effectors and bacteriocin genes, have been identified providing new clues for our understanding of pathogen virulence, aggressiveness, and host preference. These analyses will aid in efforts towards breeding for broad and durable resistance in economically important tomato and pepper cultivars.

Aldouronate utilization in Paenibacillus sp. strain JDR-2: Physiological and enzymatic evidence for coupling of extracellular depolymerization and intracellular metabolism.

Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium isolated from decaying sweet gum wood, secretes a multimodular glycohydrolase family GH10 endoxylanase (XynA1) anchored to the cell surface. The gene encoding XynA1 is part of a xylan utilization regulon that includes an aldouronate utilization gene cluster with genes encoding a GH67 alpha-glucuronidase (AguA), a GH10 endoxylanase (XynA2), and a GH43 arabinofuranosidase/beta-xylosidase (XynB). Here we show that this Paenibacillus sp. strain is able to utilize methylglucuronoxylose (MeGAX(1)), an aldobiuronate product that accumulates during acid pretreatment of lignocellulosic biomass, and methylglucuronoxylotriose (MeGAX(3)), the product of the extracellular XynA1 acting on methylglucuronoxylan (MeGAX(n)). The average rates of utilization of MeGAX(n), MeGAX(1), and MeGAX(3) were 149.8, 59.4, and 54.3 microg xylose equivalents.ml(-1).h(-1), respectively, and were proportional to the specific growth rates on the substrates. AguA was active with MeGAX(1) and MeGAX(3), releasing 4-O-methyl-d-glucuronate alpha-1,2 linked to a nonreducing terminal xylose residue. XynA2 converted xylotriose, generated by the action of AguA on MeGAX(3), to xylose and xylobiose. The ability to utilize MeGAX(1) provides a novel metabolic potential for bioconversion of acid hydrolysates of lignocellulosics. The 2.8-fold-greater rate of utilization of polymeric MeGAX(n) than that of MeGAX(3) indicates that there is coupling of extracellular depolymerization, assimilation, and intracellular metabolism, allowing utilization of lignocellulosics with minimal pretreatment. Along with adjacent genes encoding transcriptional regulators and ABC transporter proteins, the aguA and xynA2 genes in the cluster described above contribute to the efficient utilization of aldouronates derived from dilute acid and/or enzyme pretreatment protocols applied to the conversion of hemicellulose to biofuels and chemicals.

To see or not to see: infants prefer to follow the gaze of a reliable looker.

In two experiments, we examined whether 14-month-olds understand the subjective nature of gaze. In the first experiment, infants first observed an experimenter express happiness as she looked inside a container that either contained a toy (reliable looker condition) or was empty (unreliable looker condition). Then, infants had to follow the same experimenter's gaze to a target object located either behind or in front of a barrier. Infants in the reliable looker condition followed the experimenter's gaze behind the barrier more often than infants in the unreliable looker condition, whereas both groups followed the experimenter's gaze to the target object located in front of the barrier equally often. In the second experiment, infants did not generalize their knowledge about the unreliability of a looker to a second 'naïve' looker. These findings suggest that 14-month-old infants adapt their gaze following as a function of their past experience with the looker.

Structure, function, and regulation of the aldouronate utilization gene cluster from Paenibacillus sp. strain JDR-2.

Direct bacterial conversion of the hemicellulose fraction of hardwoods and crop residues to biobased products depends upon extracellular depolymerization of methylglucuronoxylan (MeGAX(n)), followed by assimilation and intracellular conversion of aldouronates and xylooligosaccharides to fermentable xylose. Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium, secretes a multimodular cell-associated GH10 endoxylanase (XynA1) that catalyzes depolymerization of MeGAX(n) and rapidly assimilates the principal products, beta-1,4-xylobiose, beta-1,4-xylotriose, and MeGAX(3), the aldotetrauronate 4-O-methylglucuronosyl-alpha-1,2-xylotriose. Genomic libraries derived from this bacterium have now allowed cloning and sequencing of a unique aldouronate utilization gene cluster comprised of genes encoding signal transduction regulatory proteins, ABC transporter proteins, and the enzymes AguA (GH67 alpha-glucuronidase), XynA2 (GH10 endoxylanase), and XynB (GH43 beta-xylosidase/alpha-arabinofuranosidase). Expression of these genes, as well as xynA1 encoding the secreted GH10 endoxylanase, is induced by growth on MeGAX(n) and repressed by glucose. Sequences in the yesN, lplA, and xynA2 genes within the cluster and in the distal xynA1 gene show significant similarity to catabolite responsive element (cre) defined in Bacillus subtilis for recognition of the catabolite control protein (CcpA) and consequential repression of catabolic regulons. The aldouronate utilization gene cluster in Paenibacillus sp. strain JDR-2 operates as a regulon, coregulated with the expression of xynA1, conferring the ability for efficient assimilation and catabolism of the aldouronate product generated by a multimodular cell surface-anchored GH10 endoxylanase. This cluster offers a desirable metabolic potential for bacterial conversion of hemicellulose fractions of hardwood and crop residues to biobased products.

Multiple-strand displacement and identification of single nucleotide polymorphisms as markers of genotypic variation of Pasteuria penetrans biotypes infecting root-knot nematodes.

Pasteuria species are endospore-forming obligate bacterial parasites of soil-inhabiting nematodes and water-inhabiting cladocerans, e.g. water fleas, and are closely related to Bacillus spp. by 16S rRNA gene sequence. As naturally occurring bacteria, biotypes of Pasteuria penetrans are attractive candidates for the biocontrol of various Meloidogyne spp. (root-knot nematodes). Failure to culture these bacteria outside their hosts has prevented isolation of genomic DNA in quantities sufficient for identification of genes associated with host recognition and virulence. We have applied multiple-strand displacement amplification (MDA) to generate DNA for comparative genomics of biotypes exhibiting different host preferences. Using the genome of Bacillus subtilis as a paradigm, MDA allowed quantitative detection and sequencing of 12 marker genes from 2000 cells. Meloidogyne spp. infected with P. penetrans P20 or B4 contained single nucleotide polymorphisms (SNPs) in the spoIIAB gene that did not change the amino acid sequence, or that substituted amino acids with similar chemical properties. Individual nematodes infected with P. penetrans P20 or B4 contained SNPs in the spoIIAB gene sequenced in MDA-generated products. Detection of SNPs in the spoIIAB gene in a nematode indicates infection by more than one genotype, supporting the need to sequence genomes of Pasteuria spp. derived from single spore isolates.

Attitudes toward videotelephones: an exploratory study of older adults with depression.

Although technology holds promising benefits to support older adults at home, attitudes can be a hindrance to the acceptance and use of new technologies. The aim of this study was to explore attitudes of older adults with depressive symptoms using videotelephones (VTs) in their homes. A descriptive design involving 4 participants who received problem solving interventions from a clinical psychologist via VTs was used. Data analysis revealed that the participants' preattitudes were dependent on their active or passive role in the learning process of the new technology. Their postattitudes were classified as ambivalent and positive. Two participants who had a positive attitude toward the VT expressed a positive behavior use.

How to adapt existing evidence-based clinical practice guidelines: a case example with smoking cessation guidelines in Canada.

OBJECTIVE: To develop and encourage the adoption of clinical practice guidelines (CPGs) for smoking cessation in Canada by engaging stakeholders in the adaptation of existing high-quality CPGs using principles of the ADAPTE framework. METHODS: An independent expert body in guideline review conducted a review and identified six existing CPGs, which met a priori criteria for quality and potential applicability to the local context. Summary statements were extracted and assigned a grade of recommendation and level of evidence by a second expert panel. Regional knowledge exchange brokers recruited additional stakeholders to build a multidisciplinary network of over 800 clinicians, researchers and decision-makers from across Canada. This interprofessional network and other stakeholders were offered various opportunities to provide input on the guideline both online and in person. We actively encouraged end-user input into the development and adaptation of the guidelines to ensure applicability to various practice settings and to promote adoption. RESULTS: The final guideline contained 24 summary statements along with supporting clinical considerations, across six topic area sections. The guideline was adopted by various provincial/territorial and national government and non-governmental organisations. CONCLUSIONS: This method can be applied in other jurisdictions to adapt existing high-quality smoking cessation CPGs to the local context and to facilitate subsequent adoption by various stakeholders.

Complete genome sequence of Paenibacillus sp. strain JDR-2.

Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium isolated from sweetgum (Liquidambar styraciflua) wood, is able to efficiently depolymerize, assimilate and metabolize 4-O-methylglucuronoxylan, the predominant structural component of hardwood hemicelluloses. A basis for this capability was first supported by the identification of genes and characterization of encoded enzymes and has been further defined by the sequencing and annotation of the complete genome, which we describe. In addition to genes implicated in the utilization of ß-1,4-xylan, genes have also been identified for the utilization of other hemicellulosic polysaccharides. The genome of Paenibacillus sp. JDR-2 contains 7,184,930 bp in a single replicon with 6,288 protein-coding and 122 RNA genes. Uniquely prominent are 874 genes encoding proteins involved in carbohydrate transport and metabolism. The prevalence and organization of these genes support a metabolic potential for bioprocessing of hemicellulose fractions derived from lignocellulosic resources.

Sequential performance in young and older adults: evidence of chunking and inhibition.

Two experiments were conducted to examine possible sources of age-related decline in sequential performance: age differences in sequence representation, retrieval of sequence elements, and efficiency of inhibitory processes. Healthy young and older participants learned a sequence of eight animal drawings in fixed order, then monitored for these targets within trials of mis-ordered stimuli, responding only when targets were shown in the correct order. Responses were slower for odd numbered targets, suggesting that participants spontaneously organized the sequence in two-element chunks. Perseverations (responses to previously relevant targets) served as an index of inhibitory inefficiency. Efficiency of chunk retrieval and self-inhibition were lower for older than for younger adults. Increasing environmental support in Experiment 2 through overt articulation of current chunk elements showed a pattern of results similar to Experiment 1, with particular benefit for older adults. The findings suggest an underlying susceptibility to interference in old age.

The effect of a looker's past reliability on infants' reasoning about beliefs.

We investigated whether 16-month-old infants' past experience with a person's gaze reliability influences their expectation about the person's ability to form beliefs. Infants were first administered a search task in which they observed an experimenter show excitement while looking inside a box that either contained a toy (reliable looker condition) or was empty (unreliable looker condition). The infants were then administered a true belief task in which they watched as the same experimenter hid a toy in 1 of 2 locations. In the test trial, the infants witnessed the experimenter search for the toy in a location that was consistent or inconsistent with her belief about the toy's location. Results for the true belief task indicated that only the infants in the reliable looker condition looked longer at the incongruent than at the congruent search behavior. These findings are consistent with evidence suggesting that infants encode the identity of agents based on past reliability and implicitly attribute beliefs to others during the 2nd year of life.

The developmental origins of naïve psychology in infancy.

Research interest in children's understanding of the mind goes back as far as Piaget's claim that children are cognitively egocentric (Flavell, 2000). Many years later, research on the understanding of the mind was revived in a paper that sought evidence for a theory of mind, not for children but for chimpanzees (Premack & Woodruff, 1978). The researchers claimed that chimpanzees' ability to predict what a human actor will do to achieve certain goals implies that the animal attributes mental states to the actor. This seminal paper generated a flurry of studies on theory of mind in nonhuman primates. A review of this research based on several different experimental paradigms concluded that chimpanzees understand others in terms of a perception-goal psychology (i.e., they can perceive what the other's goal is but not understand the mental states associated with the goal), as opposed to a full-fledged, human-like belief-desire psychology (Call & Tomasello, 2008). Around the same time, research on children's understanding of the mind was revived in a landmark paper by Wimmer and Perner (1983) and by other developmentalists (Bretherton, McNew, & Beegly-Smith. 1981). In line with the research on nonhuman primates, part of the progress that has been made in recent years is a recognition that theory of mind knowledge is acquired in an extended series of developmental milestones and that this development is based on a rich set of socio-cognitive abilities that develop in infancy (Wellman, 2002). The evidence outlined in the sections of this chapter suggests that infants possess a nascent understanding of mental states that older children use in explaining and predicting human behavior. Researchers have learned a great deal about the developmental origins of naive psychology in infancy. Nevertheless, the depth of infants' understanding of human behavior is still a controversial issue. For example, a popular paradigm in naive psychology is violation of expectancy. In false-belief tasks, infants look longer at a scene.in which a protagonist searches for an object in a location she does not know than at a scene in which the protagonist searches for an object in a location where she has previously seen the object disappear. The fact that no active behavioral response is required makes many researchers doubt that an infants' looking pattern reflects a deep level of understanding. Looking pattern may simply reflect the infants' detection that something in the scene is novel (e.g., protagonist looks at a location different than the one infants last saw her look at). Indeed this interpretation may account for the conflicting results in recent studies (e.g., Poulin-Dubois et al., 2007; Onishi & Baillargeon, 2005; Surian et al., 2007). Poulin-Dubois et al. (2007) recently reported that the ability to distinguish between knowledge and ignorance (true belief) is absent at 14 months of age and still fragile at 18 months in a violation-of-expectancy task depicting videotaped human actors. In contrast, false-belief attribution to a computer animated caterpillar has been reported in 13-month-old infants (Surian et al., 2007). Given that infants have had more experience with humans looking at objects than with a caterpillar's looking behavior, the current evidence for an implicit understanding of advanced mental states such as false belief should be interpreted with caution. As is the case for nonhuman primate research, infants' mind-reading success might be accounted for by a simple behavior-reading explanation. According to some researchers, primates' (and infants') successful performance in theory of mind tasks can be explained by a sophisticated form of behavior reading. Under this view, infants perform well in such tasks because they are adept at calculating the statistical likelihood that some aspects of people's observable features (e.g., gaze) will be linked to future actions (e.g., search at a location). Distinguishing between a mentalistic and rule-based account is very difficult (Povinelli & Vonk, 2004). One way to address this debate would be to design training studies that provide infants with first-person experience of mental states and to use more active behavioral measures. In terms of training, there is some evidence that infants' performance on goal and visual perception attribution tasks is improved if they received training of relevant skills (e.g., wearing a blindfold, reaching with a "sticky mitten": Meltzoff & Brooks, 2007: Sommerville & Woodward, 2004). Furthermore, longitudinal research using more active measures revealed links between goal detection as measured with the violation of expectancy paradigm at 10 months of age and the ability to infer intended goals in an imitation task at 14 months (Olineck & Poulin-Dubois, 2007b). Developmental changes in the scope of infants' concept of intentional agent also will require more attention from researchers. According to some, infants' attributions of intentional behavior are activated whenever infants recognize an object as a psychological agent, based on an evolutionary designed system which is sensitive to certain cues such as self-propulsion, contingent reactivity or equifinal variation of the action (Baron-Cohen, 1995; Gergely & Csibra, 2003; Johnson, 2000; Leslie, deficient in theory of mind. One may hope that nonverbal theory of mind tasks that reliably predict later theory mind skills will be adapted for use with this population and eventually used for the early detection of autism. In sum, the numerous studies reported here show that by the end of the second year of life, infants have developed ways to predict human actions The review also makes clear that we do not yet fully understand how deep infants' insight into the mind really is. Nonetheless, there appears to be some consensus that infants, like chimpanzees, understand the goals, intentions, perception, and knowledge of others. This provides the foundations for the full-fledged adult-like naive psychology that develops gradually in early childhood.