A proteomic analysis unravels novel CORVET and HOPS proteins involved in Toxoplasma gondii secretory organelles biogenesis

Apicomplexans use the endolysosomal system for the biogenesis of their secretory organelles, namely, micronemes, rhoptries, and dense granules. In Toxoplasma gondii, our previous in silico search identified the HOPS tethering but not the CORVET complex and demonstrated a role of Vps11 (a common component for both complexes) in its secretory organelle biogenesis. Herein, we performed Vps11‐GFP‐Trap pull‐down assays and identified by proteomic analysis, not only the CORVET‐specific subunit Vps8 but also a BEACH domain‐containing protein (BDCP) conserved in eukaryotes. We show that knocking‐down Vps8 affects targeting of dense granule proteins, transport of rhoptry proteins, and the localization of the cathepsin L protease vacuolar compartment marker. Only a subset of micronemal proteins are affected by the absence of Vps8, shedding light on at least two trafficking pathways involved in microneme maturation. Knocking‐down BDCP revealed a restricted and particular role of this protein in rhoptry and vacuolar compartment biogenesis. Moreover, depletion of BDCP or Vps8 abolishes parasite virulence in vivo. This study identified BDCP as a novel CORVET/HOPS‐associated protein, playing specific roles and acting in concert during secretory organelle biogenesis, an essential process for host cell infection. Our results open the hypothesis for a role of BDCP in the vesicular trafficking towards lysosome‐related organelles in mammals and yeast

Effect of pH control on lactic acid fermentation of starch by Lactobacillus manihotivorans LMG 18010T

Lactic acid fermentation of starch by #Lactobacillus manihotivorans LMG 18010T, a new amylolytic L(+) lactic acid producer, was investigated and compared with starch fermentation by #Lact. plantarum A6. At non-controlled pH, growth and lactic acid production from starch by #Lact. manihotivorans LMG 18010T lasted 25 h. Specific growth and lactic acid production rates continuously decreased from the onset of the fermentation, unlike #Lact. plantarum A6 which was able to grow and convert starch product hydrolysis into lactic acid more rapidly and efficiently at a constant rate up to pH 4.5. In spite of complete and rapid starch hydrolysis by #Lact. manihotivorans LMG 18010T during the first 6 h, only 45% of starch hydrolysis products were converted to lactic acid. When pH was maintained at 6.0, lactic acid, amylase and final biomass production by #Lact. manihotivorans LMG 18010T increased markedly and the fermentation time was reduced by half. Under the same conditions, an increase only in amylase production was observed with #Lact. plantarum A6. When grown on glucose or starch at pH 6.0, #Lact. manihotivorans LMG 18010T had an identical maximum specific growth rate (0.35/h), whereas the maximum rate of specific lactic acid production was three times higher with glucose as substrate. #Lactobacillus manihotivorans LMG 18010T did not produce amylase when grown on glucose. Based on the differences in the physiology between the two species and other amylolytic lactic acid bacteria, different applications may be expected. (Résumé d'auteur

Electrotransformation of Lactobacillus manihotivorans LMG 18010T and LMG 18011

An electroporation procedure was developed for the genetic transformation of intact cells of #Lactobacillus manihotivorans, a new #Lactobacillus species isolated from cassava sour starch fermentation in Colombia. Transformation efficiency of #Lact. manihotivorans strains LMG 18010T and LMG 18011 was measured and compared with electroporability of #Lact. plantarum strains NCIMB 8299 and LMG 9211, by investigating the effect of changes in various parameters. For #Lact. manihotivorans strain LMG 18010T, the composition of the culture medium, such as the type of peptone and the presence of Tween-80, was found to be the most critical parameter, as well as the aeration conditions of the culture ; better electroporation was obtained without air. The presence of MgCl2 in the recovery medium was favourable to regeneration of electroporated cells. Plasmid-curing of the cells did not improve their electroporability. Transformants were obtained with #Lact. manihotivorans strain LMG 18010T and the plasmids pLZ12 and pGK13, whereas #Lact. manihotivorans$ strain LMG 18011 was transformable with plasmids pLP825 and pL/Z12, with different electroporation conditions. (Résumé d'auteur

Pre-harvesting treatments to recover in a soluble form the cell-bound alpha amylase of Alicyclobacillus acidocaldarius grown in liquid culture media containing soluble and granular starch

#Alicyclobacillus acidocaldarius amylase recovery was investigated, when the strain was grown in liquid medium, containing starch as the sole carbon source. Results indicated that the amylase was non-covalently bound to the cell wall. When #A. acidocaldarius was grown with soluble as substrate, lysozyme treatment or sonication allowed the amylase release in the liquid phase. When pellets obtained from cultures grown on insolubilized starch, the lysozyme was not effective any more to release the amylase in the liquid. Nevertheless, recovery of the enzyme activity in the supernatant was possible by heating at 70°C for 5 min. It was also possible to separate the amylase directly by SDS-PAGE electrophoresis of cells and starch granule pellets, without any previous treatment. Two bands of 163 and 145 kDa (+ or - 5 kDa) respectively, presented amylolytic activity. Hydrolytic activities were found with pullulan, cyclodextrins, glycogen, amylopectin and amylose. (Résumé d'auteur

Isolation and characterization of new amylolytic strains of Lactobacillus fermentum from fermented maize doughs (mawé and ogi) from Benin

Twelve amylolytic heterofermentative lactic acid bacteria were isolated in Benin from the fermentation processes of maize sour dough, namely ogi and mawè. Discrimination of strains was performed by DNA restriction patterns and compared with carbohydrate fermentation profiles. This allowed two new amylolytic strains, Ogi E1 and Mw2, belonging to the species #Lactobacillus fermentum$, to be distinguished. Strains Ogi E1 and Mw2 presented different amylolytic activities ; amylase from strain Mw2 was more acidophilic and mesophilic than amylase produced by strain Ogi E1. (Résumé d'auteur

Isolation and characterization of new amylolytic strains of Lactobacillus fermentum from fermented maize doughs (mawé and ogi) from Benin

Twelve amylolytic heterofermentative lactic acid bacteria were isolated in Benin from the fermentation processes of maize sour dough, namely ogi and mawè. Discrimination of strains was performed by DNA restriction patterns and compared with carbohydrate fermentation profiles. This allowed two new amylolytic strains, Ogi E1 and Mw2, belonging to the species #Lactobacillus fermentum$, to be distinguished. Strains Ogi E1 and Mw2 presented different amylolytic activities ; amylase from strain Mw2 was more acidophilic and mesophilic than amylase produced by strain Ogi E1. (Résumé d'auteur