No abstract

Background: Shotgun sequences of DNA extracts from whole organisms allow a comprehensive assessment of possible symbionts. The current project makes use of four shotgun datasets from three species of the planktonic freshwater crustaceans Daphnia: one dataset from clones of D. pulex and D. pulicaria and two datasets from one clone of D. magna. We analyzed these datasets with three aims: First, we search for bacterial symbionts, which are present in all three species. Second, we search for evidence for Cyanobacteria and plastids, which had been suggested to occur as symbionts in a related Daphnia species. Third, we compare the metacommunities revealed by two different 454 pyrosequencing methods (GS 20 and GS FLX).

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, ␤-1,4-xylobiose, ␤-1,4-xylotriose, and MeGAX 3 , the aldotetrauronate 4-O-methylglucuronosyl-␣-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 ␣-glucuronidase), XynA2 (GH10 endoxylanase), and XynB (GH43 ␤-xylosidase/␣-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.

No abstract

Children's exposure to secondhand smoke (SHS) at home has numerous adverse health effects. This study evaluated the effects of a pediatric in-patient department-based pilot smoking cessation intervention for household members to reduce children's SHS exposure and encourage smoking cessation. A pre-post test design study was designed to assess the effectiveness of a telephone counseling intervention on household members of hospitalized children in pediatric departments. Data were collected with a standardized Chinese language questionnaire. At the three-month follow-up survey, the proportions of household members who reported adopting complete smoking restriction at home (55%), did not smoke at home at all (37%), did not allow others to smoke in the car (70%), or did not allow others to smoke around the child (57%) were significantly higher than the self-reported responses at the baseline survey. The proportions of household members who reported smoking at home (49%) and in the car (22%) were significantly lower than the baseline survey. Overall, 7% of the participants had reported quitting smoking after three months. Pediatric in-patient department-based telephone counseling for smoking cessation was found to be acceptable to Chinese parents. The intervention encouraged few parents to quit smoking, but encouraged more parents to take measures to reduce children's SHS exposure.

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 ␣-glucuronidase (AguA), a GH10 endoxylanase (XynA2), and a GH43 arabinofuranosidase/␤-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 g xylose equivalents ⅐ ml ؊1 ⅐ 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 ␣-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.

Overcoming the recalcitrance in lignocellulosic biomass for efficient hydrolysis of the polysaccharides cellulose and hemicellulose to fermentable sugars is a research priority for the transition from a fossilfuel-based economy to a renewable carbohydrate economy. Methylglucuronoxylans (MeGXn) are the major components of hemicellulose in woody biofuel crops. Here, we describe efficient production of the GH10 xylanase Xyl10B from Thermotoga maritima in transplastomic plants and demonstrate exceptional stability and catalytic activities of the in planta produced enzyme. Fully expanded leaves from homotransplastomic plants contained enzymatically active Xyl10B at a level of 11-15% of their total soluble protein. Transplastomic plants and their seed progeny were morphologically indistinguishable from non-transgenic plants. Catalytic activity of in planta produced Xyl10B was detected with poplar, sweetgum and birchwood xylan substrates following incubation between 40 and 90 °C and was also stable in dry and stored leaves. Optimal yields of Xyl10B were obtained from dry leaves if crude protein extraction was performed at 85 °C. The transplastomic plant derived Xyl10B showed exceptional catalytic activity and enabled the complete hydrolysis of MeGXn to fermentable sugars with the help of a single accessory enzyme (α-glucuronidase) as revealed by the sugar release assay. Even without this accessory enzyme, the majority of MeGXn was hydrolyzed by the transplastomic plant-derived Xyl10B to fermentable xylose and xylobiose.

Variable-number tandem repeats (VNTRs) have been identified in populations of Pasteuria ramosa, a castrating endobacterium of Daphnia species. The allelic polymorphisms at 14 loci in laboratory and geographically diverse soil samples showed that VNTRs may serve as biomarkers for the genetic characterization of P. ramosa isolates.