Enhanced Activity by Genetic Complementarity: Heterologous Secretion of Clostridial Cellulases by Bacillus licheniformis and Bacillus velezensis

To adapt to various ecological niches, the members of genus Bacillus display a wide spectrum of glycoside hydrolases (GH) responsible for the hydrolysis of cellulose and lignocellulose. Being abundant and renewable, cellulose-containing plant biomass may be applied as a substrate in second-generation biotechnologies for the production of platform chemicals. The present study aims to enhance the natural cellulase activity of two promising 2,3-butanediol (2,3-BD) producers, Bacillus licheniformis 24 and B. velezensis 5RB, by cloning and heterologous expression of cel8A and cel48S genes of Acetivibrio thermocellus. In B. licheniformis, the endocellulase Cel8A (GH8) was cloned to supplement the action of CelA (GH9), while in B. velezensis, the cellobiohydrolase Cel48S (GH48) successfully complemented the activity of endo-cellulase EglS (GH5). The expression of the natural and heterologous cellulase genes in both hosts was demonstrated by reverse-transcription PCR. The secretion of clostridial cellulases was additionally enhanced by enzyme fusion to the subtilisin-like signal peptide, reaching a significant increase in the cellulase activity of the cell-free supernatants. The results presented are the first to reveal the possibility of genetic complementation for enhancement of cellulase activity in bacilli, thus opening the prospect for genetic improvement of strains with an important biotechnological application.

Interdependence, Reflexivity, Fidelity, Impedance Matching, and the Evolution of Genetic Coding

ABSTRACTGenetic coding is generally thought to have required ribozymes whose functions were taken over by polypeptide aminoacyl-tRNA synthetases (aaRS). Two discoveries about aaRS and their tRNA substrates now furnish a unifying rationale for the opposite conclusion: that the key processes of the Central Dogma of molecular biology emerged simultaneously and naturally from simple origins in a peptide•RNA partnership, eliminating the epistemological need for a prior RNA world. First, the two aaRS classes likely arose from opposite strands of the same ancestral gene, implying a simple genetic alphabet. Inversion symmetries in aaRS structural biology arising from genetic complementarity would have stabilized the initial and subsequent differentiation of coding specificities and hence rapidly promoted diversity in the proteome. Second, amino acid physical chemistry maps onto tRNA identity elements, establishing reflexivity in protein aaRS. Bootstrapping of increasingly detailed coding is thus intrinsic to polypeptide aaRS, but impossible in an RNA world. These notions underline the following concepts that contradict gradual replacement of ribozymal aaRS by polypeptide aaRS: (i) any set of aaRS must be interdependent; (ii) reflexivity intrinsic to polypeptide aaRS production dynamics promotes bootstrapping; (iii) takeover of RNA-catalyzed aminoacylation by enzymes will necessarily degrade specificity; (iv) the Central Dogma’s emergence is most probable when replication and translation error rates remain comparable. These characteristics are necessary and sufficient for the essentially de novo emergence of a coupled gene-replicase-translatase system of genetic coding that would have continuously preserved the functional meaning of genetically encoded protein genes whose phylogenetic relationships match those observed today.

Female dragons, Ctenophorus pictus, do not prefer scent from unrelated males

Female choice for genetic complementarity or unrelated males occurs in several vertebrate taxa, but only a few species per higher-order taxon have so far been studied. This is particularly the case in reptiles, where female choice traditionally has been considered to be less important than in most other vertebrates. Many species of reptiles use scent marking in relation to territory use and, consequently, females may use this information to avoid settling on genetically incompatible (e.g. related) males’ territories. We tested the response of female Australian dragon lizards, Ctenophorus pictus, to pheromones from closely related and unrelated males. Contrary to predictions, females did not prefer to associate with scent from unrelated males. The reason for the lack of differential response to related and unrelated males could be explained by weak selection against inbreeding, high dependence on visual cues, female control over copulation, or post-copulatory female choice. Alternative hypotheses are discussed in relation to the biology of the species and lizard biology in general.