An intracellular membrane protein GEP1 regulates xanthurenic acid induced gametogenesis of malaria parasites.

Gametocytes differentiation to gametes (gametogenesis) within mosquitos is essential for malaria parasite transmission. Both reduction in temperature and mosquito-derived XA or elevated pH are required for triggering cGMP/PKG dependent gametogenesis. However, the parasite molecule for sensing or transducing these environmental signals to initiate gametogenesis remains unknown. Here we perform a CRISPR/Cas9-based functional screening of 59 membrane proteins expressed in the gametocytes of Plasmodium yoelii and identify that GEP1 is required for XA-stimulated gametogenesis. GEP1 disruption abolishes XA-stimulated cGMP synthesis and the subsequent signaling and cellular events, such as Ca2+ mobilization, gamete formation, and gametes egress out of erythrocytes. GEP1 interacts with GCα, a cGMP synthesizing enzyme in gametocytes. Both GEP1 and GCα are expressed in cytoplasmic puncta of both male and female gametocytes. Depletion of GCα impairs XA-stimulated gametogenesis, mimicking the defect of GEP1 disruption. The identification of GEP1 being essential for gametogenesis provides a potential new target for intervention of parasite transmission.

[Diversity of culturable sulfur-oxidizing bacteria in deep-sea hydrothermal vent environments of the South Atlantic].

OBJECTIVE: To investigate the diversity of culturable sulfur-oxidizing bacteria in hydrothermal vent environments of the South Atlantic, and analyze their characteristics of sulfur oxidation. METHODS: We enriched and isolated sulfur-oxidizing bacteria from hydrothermal vent samples collected from the South Atlantic. The microbial diversity in enrichment cultures was analyzed using the Denatural Gradient Gel Electrophoresis method. Sulfur-oxidizing characteristics of the isolates was further studied by using ion chromatography. RESULTS: A total of 48 isolates were obtained from the deep-sea hydrothermal vent samples, which belonged to 23 genera and mainly grouped into alpha-Proteobacteria (58.3%), Actinobacteria (22.9%) and gama-Proteobacteria (18.8%). Among them, the genus Thalassospira, Martelella and Microbacterium were dominant. About 60% of the isolates exibited sulfur-oxidizing ability and strain L6M1-5 had a higher sulfur oxidation rate by comparison analysis. CONCLUSION: The diversity of sulfur-oxidizing bacteria in hydrothermal environments of the South Atlantic was reported for the first time based on culture-dependent methods. The result will help understand the biogechemical process of sulfur compounds in the deep-sea hydrothermal environments.

Mameliella atlantica sp. nov., a marine bacterium of the Roseobacter clade isolated from deep-sea sediment of the South Atlantic Ocean.

A taxonomic study was carried out on strain L6M1-5(T), which was isolated from deep-sea sediment collected from the South Atlantic Ocean. The isolate was Gram-reaction-negative, oxidase-negative and catalase-weakly positive. Growth was observed in the presence of 0.5-15% (w/v) NaCl (optimum 3-5%), at 10-41 °C (optimum 28-30 °C), and pH 5.0-10.5 (optimum pH 7.0). The principal fatty acids were summed feature 8 (C18 : 1ω7c/ω6c) (84.2%), C18 : 0 (6.3%), C12 : 1 3-OH (3.2%) and C16 : 0 (2.7%). The polar lipid profile comprised phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids, two unknown phospholipids and one unknown lipid. Ubiquinone-10 was the major quinone. The G+C content of the genomic DNA was 66.0 mol %. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain L6M1-5(T) belonged to the genus Mameliella and shared 95.8% sequence similarity with Mameliella alba JLT354-W(T). The combined genotypic and phenotypic data show that strain L6M1-5(T) represents a novel species of the genus Mameliella, for which the name Mameliella. atlantica sp. nov. is proposed. The type strain is L6M1-5(T) ( = MCCC 1A07531(T) = JCM 30230(T)).

Thioclava atlantica sp. nov., isolated from deep sea sediment of the Atlantic Ocean.

A taxonomic study was carried out on strain 13D2W-2(T), which was isolated from a sulphur-oxidizing bacterial consortium, enriched by the deep-sea sediment of the Atlantic Ocean. The isolate was observed to be Gram-negative, oxidase- and catalase-positive, short rod-shaped and motile by means of a flagellum. Growth was observed at salinities from 0.5 to 12 % and at temperatures from 4 to 41 °C, and the strain found to be able to reduce nitrate but not degrade gelatin. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 13D2W-2(T) belongs to the genus Thioclava, with highest sequence similarity of 97.8 % to Thioclava dalianensis DLFJ1-1(T), followed by Thioclava pacifica TL 2(T) (97.7 %), while the sequence similarities to other members of the genus were all below 97.0 %. The digital DNA:DNA hybridization estimated values between strain 13D2W-2(T) and, respectively, T. dalianensis DLFJ1-1(T) and T. pacifica TL 2(T) were 22.6 ± 2.4 and 25.6 ± 2.4 %. The ANI values between strain 13D2W-2(T) and T. dalianensis DLFJ1-1(T) and T. pacifica TL 2(T) were 78.49 and 81.91 % respectively. The principal fatty acid identified was Summed Feature 8 (C18:1 ω7c/ω6c) (74.38 %). The isoprenoid quinone of strain 13D2W-2(T) was identified as Q10 (100 %). The major polar lipids of strain 13D2W-2(T) were found to be comprised of phosphatidylethanolamine, phosphatidylglycerol, an aminophospholipid, a glycolipid and three unknown phospholipids. The G+C content of the chromosomal DNA was determined to be 65.3 mol%. The combined genotypic and phenotypic data show that strain 13D2W-2(T) represents a novel species of the genus Thioclava, for which the name Thioclava atlantica sp. nov. is proposed, with the type strain 13D2W-2(T) ( = MCCC 1A02612(T) = LMG 27145(T)).

Efficient editing of malaria parasite genome using the CRISPR/Cas9 system.

Malaria parasites are unicellular organisms residing inside the red blood cells, and current methods for editing the parasite genes have been inefficient. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and Cas9 endonuclease-mediated genome editing) system is a new powerful technique for genome editing and has been widely employed to study gene function in various organisms. However, whether this technique can be applied to modify the genomes of malaria parasites has not been determined. In this paper, we demonstrated that Cas9 is able to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. By supplying engineered homologous repair templates, we generated targeted deletion, reporter knock-in, and nucleotide replacement in multiple parasite genes, achieving up to 100% efficiency in gene deletion and 22 to 45% efficiencies in knock-in and allelic replacement. Our results establish methodologies for introducing desired modifications in the P. yoelii genome with high efficiency and accuracy, which will greatly improve our ability to study gene function of malaria parasites. Importance: Malaria, caused by infection of Plasmodium parasites, remains a world-wide public health burden. Although the genomes of many malaria parasites have been sequenced, we still do not know the functions of approximately half of the genes in the genomes. Studying gene function has become the focus of many studies; however, editing genes in malaria parasite genomes is still inefficient. Here we designed several efficient approaches, based on the CRISPR/Cas9 system, to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. Using this system, we achieved high efficiencies in gene deletion, reporter tagging, and allelic replacement in multiple parasite genes. This technique for editing the malaria parasite genome will greatly facilitate our ability to elucidate gene function.

Thioclava dalianensis sp. nov., isolated from surface seawater.

A taxonomic study was carried out on strain DLFJ1-1(T), which was isolated from an oil-degrading bacterial consortium, enriched by the surface seawater from around the Dalian Peninsula. The isolate was Gram-reaction-negative, oxidase- and catalase-positive, short-rod-shaped and non-motile. Growth was observed at salinities from 0.5 to 15 % and at temperatures from 4 to 37 °C; the strain was unable to degrade gelatin or to reduce nitrate. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain DLFJ1-1(T) belonged to the genus Thioclava, with the highest sequence similarity (96.4 %) to Thioclava pacifica TL 2(T), while the similarities to other species examined were all below 95.0 %. The principal fatty acids were C19 : 0ω8c cyclo and summed feature 8 (C18 : 1ω7c/ω6c). The major quinone of strain DLFJ1-1(T) was Q10. The major polar lipids of strain DLFJ1-1(T) were phosphatidylethanolamine and phosphatidylglycerol. The G+C content of the chromosomal DNA was 62.5 mol%. The combined genotypic and phenotypic data show that strain DLFJ1-1(T) represents a novel species of the genus Thioclava, for which the name Thioclava dalianensis sp. nov. is proposed, with the type strain DLFJ1-1(T) ( = CGMCC 1.12325(T) = LMG 27290(T) = MCCC 1A03957(T)). An emended description of the genus Thioclava is also proposed.