Energy metabolism of bloodstream form Trypanosoma theileri

Bloodstream form Trypanosoma theileri degrades glucose to acetate (47%) and succinate (45%) and, therefore, does not solely rely on glycolysis for ATP production. This trypanosomatid does not use amino acids for energy metabolism. These results refute the prevailing hypothesis that substrate availability determines the type of energy metabolism of trypanosomatids.     

Amsterdam urban canals contain novel niches for methane-cycling microorganisms

Urbanised environments have been identified as hotspots of anthropogenic methane emissions. Especially urban aquatic ecosystems are increasingly recognised as important sources of methane. However, the microbiology behind these emissions remains unexplored. Here, we applied microcosm incubations and molecular analyses to investigate the methane-cycling community of the Amsterdam canal system in the Netherlands. The sediment methanogenic communities were dominated by Methanoregulaceae and Methanosaetaceae, with co-occurring methanotrophic Methanoperedenaceae and Methylomirabilaceae indicating the potential for anaerobic methane oxidation. Methane was readily produced after substrate amendment, suggesting an active but substrate-limited methanogenic community. Bacterial 16S rRNA gene amplicon sequencing of the sediment revealed a high relative abundance of Thermodesulfovibrionia. Canal wall biofilms showed the highest initial methanotrophic potential under oxic conditions compared to the sediment. During prolonged incubations the maximum methanotrophic rate increased to 8.08 mmol g_DW⁻¹ d⁻¹ that was concomitant with an enrichment of Methylomonadaceae bacteria. Metagenomic analysis of the canal wall biofilm lead to the recovery of a single methanotroph metagenome-assembled genome. Taxonomic analysis showed that this methanotroph belongs to the genus Methyloglobulus. Our results underline the importance of previously unidentified and specialised environmental niches at the nexus of the natural and human-impacted carbon cycle.     

Genome annotation and antimicrobial properties of Bacillus toyonensis VU‐DES13, isolated from the Folsomia candida gut

Antibiotic resistance necessitates the search for new bioactive compounds with novel mechanisms of action. Natural products derived from bacteria and fungi are widely used in the field of medicine and new environments can be explored as sources of antimicrobials. Bacteria associated with springtails have shown high inhibitory activity against pathogens. Here, we characterized a bacterial strain with high potential for antimicrobial activity, isolated from the gut of the springtail Folsomia candida Willem (Collembola: Isotomidae). The strain was characterized using the ‘analytical profile index’ and the ‘minimal inhibitory concentration’ assay to test for antibiotic resistance. Agar overlay and agar disk diffusion assays were used to test the inhibitory activity of the strain and its extract against a variety of pathogens, and reporter assays were used to investigate the mode of action. High‐performance liquid chromatography was used to analyze and fractionate the extract of bacterial culture, followed by additional assays on the fractions. The genome of the strain was screened for presence of antibiotic resistance genes and secondary metabolite gene clusters. The isolate was identified as Bacillus toyonensis Jiménez et al., but it displayed differences in metabolic profile when compared to the type species. The isolate was highly resistant to penicillin and inhibited the growth of a variety of pathogenic microorganisms. Genome analysis revealed an enrichment of resistance genes for β‐lactam antibiotics compared to the type isolate. Also, secondary metabolite clusters involved in the production of siderophores, bacteriocins, and nonribosomal peptide synthetases were identified. In conclusion, a unique Bacillus strain was isolated from the gut of F. candida, for which we provide evidence of inhibitory activity against an array of pathogens. This, coupled with high resistance to penicillin as substantiated by the presence of resistance genes, points to the potential of B. toyonensis VU‐DES13 to provide a new source of antimicrobial compounds.     

Aspartate residues of the Glu-Glu-Asp-Asp (EEDD) pore locus control selectivity and permeation of the T-type Ca(2+) channel alpha(1G)

The structural determinant of the permeation and selectivity properties of high voltage-activated (HVA) Ca(2+) channels is a locus formed by four glutamate residues (EEEE), one in each P-region of the domains I-IV of the alpha(1) subunit. We tested whether the divergent aspartate residues of the EEDD locus of low voltage-activated (LVA or T-type) Ca(2+) channels account for the distinctive permeation and selectivity features of these channels. Using the whole-cell patch-clamp technique in the HEK293 expression system, we studied the properties of the alpha(1G) T-type, the alpha(1C) L-type Ca(2+) channel subunits, and alpha(1G) pore mutants, containing aspartate-to-glutamate conversions in domain III, domain IV, or both. Three characteristic features of HVA Ca(2+) channel permeation, i.e. (a) Ba(2+) over Ca(2+) permeability, (b) Ca(2+)/Ba(2+) anomalous mole fraction effect (AMFE), and (c) high Cd(2+) sensitivity, were conferred on the domain III mutant (EEED) of alpha(1G). In contrast, the relative Ca(2+)/Ba(2+) permeability and the lack of AMFE of the alpha(1G) wild type channel were retained in the domain IV mutant (EEDE). The double mutant (EEEE) displayed AMFE and a Cd(2+) sensitivity similar to that of alpha(1C), but currents were larger in Ca(2+)- than in Ba(2+)-containing solutions. The mutation in domain III, but not that in domain IV, consistently displayed outward fluxes of monovalent cations. H(+) blocked Ca(2+) currents in all mutants more efficiently than in alpha(1G). In addition, activation curves of all mutants were displaced to more positive voltages and had a larger slope factor than in alpha(1G) wild type. We conclude that the aspartate residues of the EEDD locus of the alpha(1G) Ca(2+) channel subunit not only control its permeation properties, but also affect its activation curve. The mutation of both divergent aspartates only partially confers HVA channel permeation properties to the alpha(1G) Ca(2+) channel subunit.     

The Saccharomyces cerevisiae phosphotyrosyl phosphatase activator proteins are required for a subset of the functions disrupted by protein phosphatase 2A mutations

In Saccharomyces cerevisiae, PTPA is encoded by two genes, YPA1 and YPA2. In order to examine the biological role of PTPA as potential regulator of protein phosphatase 2A (PP2A), we compared the phenotypes of the ypaDelta mutants with these of PP2A-deficient strains. While deletion of both YPA genes is lethal, deletion of YPA1 alone results in a phenotype resembling that of PP2A-deficient strains in specific aspects such as aberrant bud morphology, abnormal actin distribution, and similar growth defects under various growth conditions. These phenotypes were even more pronounced when YPA1 was deleted in a pph21Delta genetic background. Moreover, ypaDelta mutants are hypersensitive to nocodazole and show inappropriate mitotic spindle formation as previously described for mutants in the catalytic subunit of PP2A, suggesting that Ypa, like PP2A, has a function in mitotic spindle formation. These results are consistent with an in vivo role of Ypa as a regulator of PP2A. However, unlike a PP2A-deficient strain, ypaDelta mutants do not show a G2 arrest. Therefore, Ypa does not seem to play a role in the regulation of PP2A at this stage of the cell cycle. These results imply that Ypa regulates a specific subset of PP2A functions, possibly by controlling the subunit composition of PP2A.     

Phenotypic alterations in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> plants caused by<Emphasis Type="Italic"> Rhodococcus fascians</Emphasis> infection

Arabidopsis thaliana (L.) Heynh. plants were challenged with Rhodococcus fascians at several developmental stages and using different inoculation procedures. A variety of morphological alterations was scored on the infected plants; some of them resembled phenotypes of A. thaliana mutants in their shoot apical meristem (SAM) organization. Infection with R. fascians did not affect SAM organization in wild type nor in SAM mutants. Anatomical studies on the new organs formed after infection with R. fascians demonstrated extensive bacterial colonization. Colonization and concomitant production of specific signals are the likely cause of malformations.     

Factors influencing glycosylation of Trichoderma reesei cellulases. II: N-glycosylation of Cel7A core protein isolated from different strains

A systematic analysis of the N-glycosylation of the catalytic domain of cellobiohydrolase I (Cel7A or CBH I) isolated from several Trichoderma reesei strains grown in minimal media was performed. Using a combination of chromatographic, electrophoretic, and mass spectrometric methods, the presence of glucosylated and phosphorylated oligosaccharides on the three N-glycosylation sites of Cel7A core protein (from T. reesei strains Rut-C30 and RL-P37) confirms previous findings. With N-glycans isolated from other strains, no end-capping glucose could be detected. Phosphodiester linkages were however found in proteins from each strain and these probably occur on both the alpha1-3 and the alpha1-6 branch of the high-mannose oligosaccharide tree. Evidence is also presented for the occurrence of mannobiosyl units on the phosphodiester linkage. Therefore the predominant N-glycans on Cel7A can be represented as (ManP)(0-1)GlcMan(7-8)GlcNAc2 for the hyperproducing Rut-C30 and RL-P37 mutants and as (Man(1-2)P)(0-1-2)Man(5-6-7)GlcNAc2 for the wild-type strain and the other mutants. As shown by ESI-MS, random substitution of these structures on the N-glycosylation sites explains the heterogeneous glycoform population of the isolated core domains. PAG-IEF separates up to five isoforms, resulting from posttranslational modification of Cel7A with mannosyl phosphodiester residues at the three distinct sites. This study clearly shows that posttranslational phosphorylation of glycoproteins is not atypical for Trichoderma sp. and that, in the case of the Rut-C30 and RL-P37 strains, the presence of an end-capped glucose residue at the alpha1-3 branch apparently hinders a second mannophoshoryl transfer.     

TRPV channels and modulation by hepatocyte growth factor/scatter factor in human hepatoblastoma (HepG2) cells

Using patch clamp and Ca(2+) imaging techniques, we have studied Ca(2+) entry pathways in human hepatoblastoma (HepG2) cells. These cells express the mRNA of TRPV1, TRPV2, TRPV3 and TRPV4 channels, but not those of TRPV5 and TRPV6. Functional assessment showed that capsaicin (10 microM), 4alpha-phorbol-12,13-didecanoate (4alphaPDD, 1 microM), arachidonic acid (10 microM), hypotonic stress, and heat all stimulated increases in [Ca(2+)](i) within minutes. The increase in [Ca(2+)](i) depended on extracellular Ca(2+) and on the transmembrane potential, which indicated that both driving forces affected Ca(2+) entry. Capsaicin also stimulated an increase in [Ca(2+)](i) in nominally Ca(2+)-free solutions, which was compatible with the receptor functioning as a Ca(2+) release channel. Hepatocyte growth factor/scatter factor (HGF/SF) modulated Ca(2+) entry. Ca(2+) influx was greater in HepG2 cells incubated with HGF/SF (20 ng/ml for 20 h) compared with non-stimulated cells, but this occurred only in those cells with a migrating phenotype as determined by presence of a lamellipodium and trailing footplate. The effect of capsaicin on [Ca(2+)](i) was greater in migrating HGF/SF-treated cells, and this was inhibited by capsazepine. The difference between control and HGF/SF-treated cells was not found in Ca(2+)-free solutions. 4alphaPDD also had no greater effect on HGF/SF-treated cells. We conclude that TRPV1 and TRPV4 channels provide Ca(2+) entry pathways in HepG2 cells. HGF/SF increases Ca(2+) entry via TRPV1, but not via TRPV4. This rise in [Ca(2+)](i) may constitute an early response of a signalling cascade that gives rise to cell locomotion and the migratory phenotype.