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Symbioses between plants and microorganims have been fundamental in the evolution of both groups. The endophytic bacteria associated with conifers have been poorly studied in terms of diversity, ecology, and function. Coniferous trees of the genera Larix, Pseudotsugae, Picea and mainly Pinus, are hosts of many insects, including bark beetles and especially the Dendroctonus species. These insects colonize and kill these trees during their life cycle. Several bacteria detected in the gut and cuticle of these insects have been identified as endophytes in conifers. In this study, we characterized and compared the endophytic bacterial diversity in roots, phloem and bark of non-attacked saplings of Pinus arizonica and P. durangensis using 16S rRNA gene pyrosequencing. In addition, we evaluated the degree of taxonomic relatedness, and the association of metabolic function profiles of communities of endophytic bacteria and previously reported gut bacterial communities of D. rhizophagus; a specialized bark beetle that colonizes and kills saplings of these pine species. Our results showed that both pine species share a similar endophytic community. A total of seven bacterial phyla, 14 classes, 26 orders, 43 families, and 51 genera were identified. Enterobacteriaceae was the most abundant family across all samples, followed by Acetobacteraceae and Acidobacteriaceae, which agree with previous studies performed in other pines and conifers. Endophytic communities and that of the insect gut were significantly different, however, the taxonomic relatedness of certain bacterial genera of pines and insect assemblages suggested that some bacteria from pine tissues might be the same as those in the insect gut. Lastly, the metabolic profile using PICRUSt showed there to be a positive association between communities of both pines and insect gut. This study represents the baseline into the knowledge of the endophytic bacterial communities of two of the major hosts affected by D. rhizophagus.

Here, we describe a zymographic method for the simultaneous detection of enzymatic activity and molecular weight (MW) estimation, following a single electrophoresis step. This involved separating cellulase and xylanase activities from bacteria and fungi, obtained from different sources, such as commercial extracts, crude extract and purified proteins, under denaturing conditions, by 10% polyacrylamide gel electrophoresis, using polyacrylamide gels copolymerized with 1% (w/v) carboxymethylcellulose or beechwood xylan as substrates. Then, enzymes were refolded by treatment with 2.5% Triton X-100 in an appropriate buffer for each enzymatic activity, and visualized by Coomassie blue staining for MW estimation. Finally, Congo red staining revealed bio-active cellulase and xylanase bands after electrophoretic separation of the proteins in the preparations. This method may provide a useful additional tool for screening of particular cellulase and xylanase producers, identification and MW estimation of polypeptides that manifest these activities, and for monitoring and control of fungal and bacterial cellulase and xylanase production.

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Rahnella sp. ChDrAdgB13 is a dominant member of the gut bacterial core of species of the genus Dendroctonus, which is one of the most destructive pine forest bark beetles. The objectives of this study were identified in Rahnella sp. ChDrAdgB13 genome the glycosyl hydrolase families involved in carbohydrate metabolism and specifically, the genes that participate in xylan hydrolysis, to determine the functionality of a putative endo-1,4-β-D-xylanase, which results to be bifunctional xylanase–ferulic acid esterase called R13 Fae and characterize it biochemically. The carbohydrate-active enzyme prediction revealed 25 glycoside hydrolases, 20 glycosyl transferases, carbohydrate esterases, two auxiliary activities, one polysaccharide lyase, and one carbohydrate-binding module (CBM). The R13 Fae predicted showed high identity to the putative esterases and glycosyl hydrolases from Rahnella species and some members of the Yersiniaceae family. The r13 fae gene encodes 393 amino acids (43.5 kDa), containing a signal peptide, esterase catalytic domain, and CBM48. The R13 Fae modeling showed a higher binding affinity to ferulic acid, α-naphthyl acetate, and arabinoxylan, and a low affinity to starch. The R13 Fae recombinant protein showed activity on α-naphthyl acetate and xylan, but not on starch. This enzyme showed mesophilic characteristics, displaying its optimal activity at pH 6.0 and 25°C. The enzyme was stable at pH from 4.5 to 9.0, retaining nearly 66–71% of its original activity. The half-life of the enzyme was 23 days at 25°C. The enzyme was stable in the presence of metallic ions, except for Hg2+. The products of R13 Fae mediated hydrolysis of beechwood xylan were xylobiose and xylose, manifesting an exo-activity. The results suggest that Rahnella sp. ChDrAdgB13 hydrolyze xylan and its products could be assimilated by its host and other gut microbes as a nutritional source, demonstrating their functional role in the bacterial-insect interaction contributing to their fitness, development, and survival.

Dendroctonus bark beetles (Scolytinae) are one of the most important disturbance agents of coniferous forests in North and Central America. These beetles spend their lives almost entirely under the tree bark, and their survival and reproductive success depend on their ability to overcome the toxic effect of the trees’ oleoresin. The cytochromes P450 (CYPs) are associated with the detoxification process of xenobiotics, as well as other physiological processes. Different cytochromes (families 4, 6, and 9) in the Dendroctonus species have been expressed under several experimental conditions; nevertheless, the expression time-course of these genes is unknown. To explore the induction speed of CYPs , we evaluated the relative expression of the CYP6BW5 , CYP6DG1 , CYP6DJ2 , CYP9Z18 , and CYP9Z20 genes at the early hours of drilling and settling into a tree (1, 2, 4, 6, 8, 12, 18 h) both in females and males, solitary or paired, of the bark beetle Dendroctonus rhizophagus Thomas and Bright. Our findings show that the five genes were rapidly overexpressed in the early hours (1 to 6 h) in both sexes and in solitary and paired conditions, suggesting their participation in the detoxification process. Additionally, the CYPs expression shows up- and down-regulation patterns through these short times, suggesting their probable participation in other physiological processes as the biosynthesis of hormones, pheromones or compounds related to reproduction.

Species belonging to the genus Rahnella are dominant members of the core gut bacteriome of Dendroctonus-bark beetles, a group of insects that includes the most destructive agents of pine forest in North and Central America, and Eurasia. From 300 isolates recovered from the gut of these beetles, 10 were selected to describe an ecotype of Rahnella contaminans. The polyphasic approach conducted with these isolates included phenotypic characteristics, fatty acid analysis, 16S rRNA gene, multilocus sequence analyses (gyrB, rpoB, infB, and atpD genes), and complete genome sequencing of two isolates, ChDrAdgB13 and JaDmexAd06, representative of the studied set. Phenotypic characterization, chemotaxonomic analysis, phylogenetic analyses of the 16S rRNA gene, and multilocus sequence analysis showed that these isolates belonged to Rahnella contaminans. The G + C content of the genome of ChDrAdgB13 (52.8%) and JaDmexAd06 (52.9%) was similar to those from other Rahnella species. The ANI between ChdrAdgB13 and JaDmexAd06 and Rahnella species including R. contaminans, varied from 84.02 to 99.18%. The phylogenomic analysis showed that both strains integrated a consistent and well-defined cluster, together with R. contaminans. A noteworthy observation is the presence of peritrichous flagella and fimbriae in the strains ChDrAdgB13 and JaDmexAd06. The in silico analysis of genes encoding the flagellar system of these strains and Rahnella species showed the presence of flag-1 primary system encoding peritrichous flagella, as well as fimbriae genes from the families type 1, α, β and σ mainly encoding chaperone/usher fimbriae and other uncharacterized families. All this evidence indicates that isolates from the gut of Dendroctonus-bark beetles are an ecotype of R. contaminans, which is dominant and persistent in all developmental stages of these bark beetles and one of the main members of their core gut bacteriome.