Cultivation of marine bacteria of the SAR202 clade.

Bacteria of the SAR202 clade, within the phylum Chloroflexota, are ubiquitously distributed in the ocean but have not yet been cultivated in the lab. It has been proposed that ancient expansions of catabolic enzyme paralogs broadened the spectrum of organic compounds that SAR202 bacteria could oxidize, leading to transformations of the Earth's carbon cycle. Here, we report the successful cultivation of SAR202 bacteria from surface seawater using dilution-to-extinction culturing. The growth of these strains is very slow (0.18-0.24 day-1) and is inhibited by exposure to light. The genomes, of ca. 3.08 Mbp, encode archaella (archaeal motility structures) and multiple sets of enzyme paralogs, including 80 genes coding for enolase superfamily enzymes and 44 genes encoding NAD(P)-dependent dehydrogenases. We propose that these enzyme paralogs participate in multiple parallel pathways for non-phosphorylative catabolism of sugars and sugar acids. Indeed, we demonstrate that SAR202 strains can utilize several substrates that are metabolized through the predicted pathways, such as sugars ʟ-fucose and ʟ-rhamnose, as well as their lactone and acid forms.

Culturing the ubiquitous freshwater actinobacterial acI lineage by supplying a biochemical 'helper' catalase.

The actinobacterial acI lineage is among the most successful and ubiquitous freshwater bacterioplankton found on all continents, often representing more than half of all microbial cells in the lacustrine environment and constituting multiple ecotypes. However, stably growing pure cultures of the acI lineage have not been established despite various cultivation efforts based on ecological and genomic studies on the lineage, which is in contrast to the ocean from which abundant microorganisms such as Prochlorococcus, Pelagibacter, and Nitrosopumilus have been isolated. Here, we report the first two pure cultures of the acI lineage successfully maintained by supplementing the growth media with catalase. Catalase was critical for stabilizing the growth of acI strains irrespective of the genomic presence of the catalase-peroxidase (katG) gene. The two strains, representing two novel species, displayed differential phenotypes and distinct preferences for reduced sulfurs and carbohydrates, some of which were difficult to predict based on genomic information. Our results suggest that culture of previously uncultured freshwater bacteria can be facilitated by a simple catalase-supplement method and indicate that genome-based metabolic prediction can be complemented by physiological analyses.

Methylglyoxal upregulates Dictyostelium discoideum slug migration by triggering glutathione reductase and methylglyoxal reductase activity.

Glutathione (GSH)-deprived Dictyostelium discoideum accumulates methylglyoxal (MG) and reactive oxygen species (ROS) during vegetative growth. However, the reciprocal effects of the production and regulation of these metabolites on differentiation and cell motility are unclear. Based on the inhibitory effects of γ-glutamylcysteine synthetase (gcsA) disruption and GSH reductase (gsr) overexpression on aggregation and culmination, respectively, we overexpressed GSH-related genes encoding superoxide dismutase (Sod2), catalase (CatA), and Gcs, in D. discoideum. Wild-type KAx3 and gcsA-overexpressing (gcsAOE) slugs maintained GSH levels at levels of approximately 2.1-fold less than the reference GSH synthetase-overexpressing mutant; their GSH levels did not correlate with slug migration ability. Through prolonged KAx3 migration by treatment with MG and H2O2, we found that MG increased after the mound stage in this strain, with a 2.6-fold increase compared to early developmental stages; in contrast, ROS were maintained at high levels throughout development. While the migration-defective sod2- and catA-overexpressing mutant slugs (sod2OE and catAOE) decreased ROS levels by 50% and 53%, respectively, these slugs showed moderately decreased MG levels (36.2±5.8 and 40.7±1.6nmolg-1 cells wet weight, P<0.05) compared to the parental strain (54.2±3.5nmolg-1). Importantly, defects in the migration of gcsAOE slugs decreased MG considerably (13.8±4.2nmolg-1, P<0.01) along with a slight decrease in ROS. In contrast to the increase observed in migrating sod2OE and catAOE slugs by treatment with MG and H2O2, the migration of gcsAOE slugs appeared unaffected. This behavior was caused by MG-triggered Gsr and NADPH-linked aldolase reductase activity, suggesting that GSH biosynthesis in gcsAOE slugs is specifically used for MG-scavenging activity. This is the first report showing that MG upregulates slug migration via MG-scavenging-mediated differentiation.

Characterization of spatial distribution of the bacterial community in the South Sea of Korea.

In order to investigate the importance of spatial and environmental factors on the structure and diversity of bacterial communities, high-resolution 16S rRNA gene tag pyrosequencing was applied to bacterial communities in the littoral sea. Seawater samples were prepared from seven different stations in the South Sea of Korea, the marginal sea in the western Pacific Ocean, and were divided into three groups according to distances from the coastline. The majority of 19,860 sequences were affiliated with Alphaproteobacteria (58.2%), Gammaproteobacteria (7.9%), and Bacteroidetes (13.9%). The bacterioplankton community at each station was highly diverse and varied among the samples. Major bacterial lineages showed different niche preferences among three locational groups. Alphaproteobacteria was the most abundant bacterial class, and it harbored the most frequently recorded operational taxonomic units (OTUs) in all sampling stations. However, dominant groups at the order levels showed a clear difference among the samples. The SAR11 clade was more abundant in coastal waters while the Roseobacter clade prevailed at stations far away from the coastline. Furthermore, members of Actinobacteria and Cyanobacteria also exhibited spatial variability. The OM1 clade in Actinobacteria constituted a predominant fraction in coastal samples, but it was essentially absent at the distal stations closer to open ocean. In contrast, Synechococcus was the predominant taxon in the distal samples, accounting for 7.1-19.5%, but was hardly detected in coastal waters, representing less than 0.7%. In Bacteroidetes, NS5 and NS9 groups tended to inhabit coastal waters while the genera Polaribacter and Ulvibacter were more abundant in distal stations. Clustering analysis and principle coordinates analysis based on OTU data indicated that bacterial communities in the studied area were separated into three groups that coincided with locational grouping. Statistical analysis showed that phosphate and dissolved oxygen concentration had a significant influence on the bacterial community composition.

Translationally Controlled Tumor Protein Stimulates Dopamine Release from PC12 Cells via Ca2+-Independent Phospholipase A2 Pathways.

The translationally controlled tumor protein (TCTP), initially identified as a tumor- and growth-related protein, is also known as a histamine-releasing factor (HRF). TCTP is widely distributed in the neuronal systems, but its function is largely uncharacterized. Here, we report a novel function of TCTP in the neurotransmitter release from a neurosecretory, pheochromocytoma (PC12) cells. Treatment with recombinant TCTP (rTCTP) enhanced both basal and depolarization (50 mM KCl)-evoked [³H]dopamine release in concentration- and time-dependent manners. Interestingly, even though rTCTP induced the increase in intracellular calcium levels ([Ca2+]i), the rTCTP-driven effect on dopamine release was mediated by a Ca2+-independent pathway, as evidenced by the fact that Ca2+-modulating agents such as Ca2+ chelators and a voltage-gated L-type Ca2+-channel blocker did not produce any changes in rTCTP-evoked dopamine release. In a study to investigate the involvement of phospholipase A2 (PLA2) in rTCTP-induced dopamine release, the inhibitor for Ca2+-independent PLA2 (iPLA2) produced a significant inhibitory effect on rTCTP-induced dopamine release, whereas this release was not significantly inhibited by Ca2+-dependent cytosolic PLA2 (cPLA2) and secretory PLA2 (sPLA2) inhibitors. We found that rTCTP-induced dopamine release from neuronal PC12 cells was modulated by a Ca2+-independent mechanism that involved PLA2 in the process, suggesting the regulatory role of TCTP in the neuronal functions.

Glutathione initiates the development of Dictyostelium discoideum through the regulation of YakA.

Reduced glutathione (GSH) is an essential metabolite that performs multiple indispensable roles during the development of Dictyostelium. We show here that disruption of the gene (gcsA-) encoding y-glutamylcysteine synthetase, an essential enzyme in GSH biosynthesis, inhibited aggregation, and that this developmental defect was rescued by exogenous GSH, but not by other thiols or antioxidants. In GSH-depleted gcsA- cells, the expression ofa growth-stage-specific gene (cprD) was not inhibited, and we did not detect the expression of genes that encode proteins required for early development (cAMP receptor, carA/cAR1; adenylyl cyclase, acaA/ACA; and the catalytic subunit of protein kinase A, pkaC/PKA-C). The defects in gcsA cells were not restored by cAMP stimulation or by cAR1 expression. Further, the expression of yakA, which initiates development and induces the expression of PKA-C, ACA, and cAR1, was regulated by the intracellular concentration of GSH. Constitutive expression of YakA in gcsA- cells (YakA(OE)/gcsA-) rescued the defects in developmental initiation and the expression of early developmental genes in the absence of GSH. Taken together, these findings suggest that GSH plays an essential role in the transition from growth to development by modulating the expression of the genes encoding YakA as well as components thatact downstream in the YakA signaling pathway.

Homeoprotein Hbx4 represses the expression of the adhesion molecule DdCAD-1 governing cytokinesis and development.

We investigated the function of homeodomain-containing protein Hbx4 in Dictyostelium discoideum. Hbx4-overexpressing cells (Hbx4(OE)) displayed defects in growth rate and cytokinesis and showed differences in slug motility and cell-type proportioning from KAx3. Furthermore, the overexpression of Hbx4 inhibited the induction of cadA, which encoded the Ca(2+)-dependent cell adhesion molecule DdCAD-1, despite expression of csaA and gpaB. The electrophoretic mobility shift assay showed that the promoter of cadA contained the Hbx4-binding site. Moreover, constitutively expressed DdCAD-1 in Hbx4(OE) rescued the defects in cytokinesis and development. These results suggest that Hbx4 modulates DdCAD-1-mediated cytokinesis and cell-type proportioning.