Adsorption of beta-galactosidase of Alicyclobacillus acidocaldarius on wild type and mutants spores of Bacillus subtilis

ABSTRACT: BACKGROUND: The Bacillus subtilis spore has long been used as a surface display system with potential applications in a variety of fields ranging from mucosal vaccine delivery, bioremediation and biocatalyst development. More recently, a non-recombinant approach of spore display has been proposed and heterologous proteins adsorbed on the spore surface. We used the well-characterized beta-galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius as a model to study enzyme adsorption, to analyze whether and how spore-adsorption affects the properties of the enzyme and to improve the efficiency of the process. RESULTS: We report that purified beta-galactosidase molecules were adsorbed to purified spores of a wild type strain of B. subtilis retaining ca. 50% of their enzymatic activity. Optimal pH and temperature of the enzyme were not altered by the presence of the spore, that protected the adsorbed beta-galactosidase from exposure to acidic pH conditions. A collection of mutant strains of B. subtilis lacking a single or several spore coat proteins was compared to the isogenic parental strain for the adsorption efficiency. Mutants with an altered outermost spore layer (crust) were able to adsorb 60-80% of the enzyme, while mutants with a severely altered or totally lacking outer coat adsorbed 100% of the beta-galactosidase molecules present in the adsorption reaction. CONCLUSION: Our results indicate that the spore surface structures, the crust and the outer coat layer, have an negative effect on the adhesion of the beta-galactosidase. Electrostatic forces, previously suggested as main determinants of spore adsorption, do not seem to play an essential role in the spore-beta-galactosidase interaction. The analysis of mutants with altered spore surface has shown that the process of spore adsorption can be improved and has suggested that such improvement has to be based on a better understanding of the spore surface structure. Although the molecular details of spore adsorption have not been fully elucidated, the efficiency of the process and the pH-stability of the adsorbed molecules, together with the well documented robustness and safety of spores of B. subtilis, propose the spore as a novel, non-recombinant system for enzyme display.     

Interspecific extrinsic and intrinsic competitive interactions in egg parasitoids

Interspecific competitive interactions can occur either between adult parasitoids searching/exploiting hosts (extrinsic competition) or between parasitoid larvae developing within the same host (intrinsic competition). Understanding how interspecific competition between parasitoids can affect pest suppression is important for improving biological pest control. The purpose of this work was to review both extrinsic and intrinsic competition between egg parasitoid species. These are organisms that are often candidates for biological control programs due to their ability to kill the pest before the crop feeding stage. We first reviewed the literature about interspecific competitive abilities of adult parasitoids in terms of comparative host location strategies highlighting which ecological and behavioral factors are likely to shape extrinsic competition. Then we focused on the interspecific competitive interactions between immatures developing within the same host taking into account which factors play a key role in the outcome of intrinsic competition. Finally we conclude stressing on the need to elucidate the overall competitive interaction that parasitoid species may experience in the field in order to enhance biological control success.     

Isolation and characterization of microsatellite markers from Hibiscus rosa-sinensis (Malvaceae) and cross-species amplifications

We report on the development of 10 microsatellite markers in Hibiscus rosa-sinensis (Hrs). Three markers were obtained from sequences available in GenBank and seven were isolated using a two-step ‘primer extension’ procedure, based on the microsatellite-AFLP (M-AFLP) technique. Polymorphism was explored in 21 Hrs genotypes representing the genetic variation within commercial varieties. Inter-specific amplification was assessed on 12 Hibiscus wild species. A total of 45 and 56 alleles (ranging from 1 to 10 for each locus) was amplified respectively from the 21 Hrs varieties and among the full Hibiscus spp. genotype set. Primers and conditions for polymerase chain reaction (PCR) amplification of the detected loci are reported.     

Revealing the genetic traits of the foodborne microbial genus Hafnia: Implications for the human gut microbiome

The bacterial genus Hafnia has recently attracted attention due to its complex metabolic features and host-interaction capabilities, which are associated with health benefits, primarily weight loss. However, significant gaps remain in our understanding of the genomic characteristics of this emerging microbial group. In this study, we utilized all available high-quality genomes of Hafnia alvei and Hafnia paralvei to uncover the broad distribution of Hafnia in human and honeybee guts, as well as in dairy products, by analysing 1068 metagenomic datasets. We then investigated the genetic traits related to Hafnia's production of vitamins and short-chain fatty acids (SCFAs) through a comparative genomics analysis that included all dominant bacterial species in the three environments under study. Our findings underscore the extensive metabolic capabilities of Hafnia, particularly in the production of vitamins such as thiamine (B1), nicotinate (B3), pyridoxine (B6), biotin (B7), folate (B9), cobalamin (B12), and menaquinone (K2). Additionally, Hafnia demonstrated a conserved genetic makeup associated with SCFA production, including acetate, propanoate, and butanoate. These metabolic traits were further confirmed using RNAseq analyses of a newly isolated H. paralvei strain T10. Overall, our study illuminates the ecological distribution and genetic attributes of this bacterial genus, which is of increasing scientific and industrial relevance.     

Vitamin A deficiency alters the bioelectric parameters and RNA content of rat gastric mucosa in vitro.

The present study was aimed to investigate the mechanisms by which vitamin A plays a role in maintaining the efficiency of gastric mucosal barrier. Particularly, we measured electrical parameters and the RNA/DNA ratio of gastric mucosa isolated in vitro from the stomach of rats in which vitamin A-deficiency was induced by means of a vitamin A-free diet and then abolished by means of a massive vitamin A supplementation. Pair-fed vitamin A-nondepleted rats and normal rats fed ad libitum on a standard diet served as controls. Vitamin A status was assayed for each group of rats by measuring the hepatic content of vitamin A. We found that in gastric mucosa vitamin A-deficiency induced: 1) a decrease in both transmucosal potential difference and short-circuit current; 2) an increase in transmucosal electrical resistance; 3) a decrease in RNA content resulting in a decreased RNA/DNA ratio. Abolishment of vitamin A-deficiency restored both electrical parameters and RNA content of rat gastric mucosa. Our results stress the role of vitamin A in maintaining the efficiency of the gastric mucosal barrier. Vitamin A seems to act by stabilizing gastric electrical parameters and by controlling the protein synthesis/turnover in the surface gastric mucosal cells.