Genomic Insights into Moderately Thermophilic Methanotrophs of the Genus Methylocaldum.

Considering the increasing interest in understanding the biotic component of methane removal from our atmosphere, it becomes essential to study the physiological characteristics and genomic potential of methanotroph isolates, especially their traits allowing them to adapt to elevated growth temperatures. The genetic signatures of Methylocaldum species have been detected in many terrestrial and aquatic ecosystems. A small set of representatives of this genus has been isolated and maintained in culture. The genus is commonly described as moderately thermophilic, with the growth optimum reaching 50 °C for some strains. Here, we present a comparative analysis of genomes of three Methylocaldum strains-two terrestrial M. szegediense strains (O-12 and Norfolk) and one marine strain, Methylocaldum marinum (S8). The examination of the core genome inventory of this genus uncovers significant redundancy in primary metabolic pathways, including the machinery for methane oxidation (numerous copies of pmo genes) and methanol oxidation (duplications of mxaF, xoxF1-5 genes), three pathways for one-carbon (C1) assimilation, and two methods of carbon storage (glycogen and polyhydroxyalkanoates). We also investigate the genetics of melanin production pathways as a key feature of the genus.

Draft Genome Sequences of Two Bacteria from the Roseobacter Group.

Here, we report the draft genome sequences of strains HS012 and HS039, which were isolated from cnidarian polyps that had recently undergone metamorphosis. Genomic analyses place these strains within the Phaeobacter and Leisingera genera, members of the Roseobacter group.

The Influence of Bacteria on Animal Metamorphosis.

The swimming larvae of many marine animals identify a location on the seafloor to settle and undergo metamorphosis based on the presence of specific surface-bound bacteria. While bacteria-stimulated metamorphosis underpins processes such as the fouling of ship hulls, animal development in aquaculture, and the recruitment of new animals to coral reef ecosystems, little is known about the mechanisms governing this microbe-animal interaction. Here we review what is known and what we hope to learn about how bacteria and the factors they produce stimulate animal metamorphosis. With a few emerging model systems, including the tubeworm Hydroides elegans, corals, and the hydrozoan Hydractinia, we have begun to identify bacterial cues that stimulate animal metamorphosis and test hypotheses addressing their mechanisms of action. By understanding the mechanisms by which bacteria promote animal metamorphosis, we begin to illustrate how, and explore why, the developmental decision of metamorphosis relies on cues from environmental bacteria.

Genetic examination of the marine bacterium Pseudoalteromonas luteoviolacea and effects of its metamorphosis-inducing factors.

Pseudoalteromonas luteoviolacea is a globally distributed marine bacterium that stimulates the metamorphosis of marine animal larvae, an important bacteria-animal interaction that can promote the recruitment of animals to benthic ecosystems. Recently, different P. luteoviolacea isolates have been shown to produce two stimulatory factors that can induce tubeworm and coral metamorphosis; Metamorphosis-Associated Contractile structures (MACs) and tetrabromopyrrole (TBP) respectively. However, it remains unclear what proportion of P. luteoviolacea isolates possess the genes encoding MACs, and what phenotypic effect MACs and TBP have on other larval species. Here, we show that 9 of 19 sequenced P. luteoviolacea genomes genetically encode both MACs and TBP. While P. luteoviolacea biofilms producing MACs stimulate the metamorphosis of the tubeworm Hydroides elegans, TBP biosynthesis genes had no effect under the conditions tested. Although MACs are lethal to larvae of the cnidarian Hydractinia symbiologicarpus, P. luteoviolacea mutants unable to produce MACs are capable of stimulating metamorphosis. Our findings reveal a hidden complexity of interactions between a single bacterial species, the factors it produces and two species of larvae belonging to different phyla.

Distinctive Archaeal Composition of an Artisanal Crystallizer Pond and Functional Insights Into Salt-Saturated Hypersaline Environment Adaptation.

Hypersaline environments represent some of the most challenging settings for life on Earth. Extremely halophilic microorganisms have been selected to colonize and thrive in these extreme environments by virtue of a broad spectrum of adaptations to counter high salinity and osmotic stress. Although there is substantial data on microbial taxonomic diversity in these challenging ecosystems and their primary osmoadaptation mechanisms, less is known about how hypersaline environments shape the genomes of microbial inhabitants at the functional level. In this study, we analyzed the microbial communities in five ponds along the discontinuous salinity gradient from brackish to salt-saturated environments and sequenced the metagenome of the salt (halite) precipitation pond in the artisanal Cáhuil Solar Saltern system. We combined field measurements with spectrophotometric pigment analysis and flow cytometry to characterize the microbial ecology of the pond ecosystems, including primary producers and applied metagenomic sequencing for analysis of archaeal and bacterial taxonomic diversity of the salt crystallizer harvest pond. Comparative metagenomic analysis of the Cáhuil salt crystallizer pond against microbial communities from other salt-saturated aquatic environments revealed a dominance of the archaeal genus Halorubrum and showed an unexpectedly low abundance of Haloquadratum in the Cáhuil system. Functional comparison of 26 hypersaline microbial metagenomes revealed a high proportion of sequences associated with nucleotide excision repair, helicases, replication and restriction-methylation systems in all of them. Moreover, we found distinctive functional signatures between the microbial communities from salt-saturated (>30% [w/v] total salinity) compared to sub-saturated hypersaline environments mainly due to a higher representation of sequences related to replication, recombination and DNA repair in the former. The current study expands our understanding of the diversity and distribution of halophilic microbial populations inhabiting salt-saturated habitats and the functional attributes that sustain them.

Imaging of Cellular Oxidoreductase Activity Suggests Mixotrophic Metabolisms in Thiomargarita spp.

The largest known bacteria, Thiomargarita spp., have yet to be isolated in pure culture, but their large size allows for individual cells to be monitored in time course experiments or to be individually sorted for omics-based investigations. Here we investigated the metabolism of individual cells of Thiomargarita spp. by using a novel application of a tetrazolium-based dye that measures oxidoreductase activity. When coupled with microscopy, staining of the cells with a tetrazolium-formazan dye allows metabolic responses in Thiomargarita spp. to be to be tracked in the absence of observable cell division. Additionally, the metabolic activity of Thiomargarita sp. cells can be differentiated from the metabolism of other microbes in specimens that contain adherent bacteria. The results of our redox dye-based assay suggest that Thiomargarita is the most metabolically versatile under anoxic conditions, where it appears to express cellular oxidoreductase activity in response to the electron donors succinate, acetate, citrate, formate, thiosulfate, H2, and H2S. Under hypoxic conditions, formazan staining results suggest the metabolism of succinate and likely acetate, citrate, and H2S. Cells incubated under oxic conditions showed the weakest formazan staining response, and then only to H2S, citrate, and perhaps succinate. These results provide experimental validation of recent genomic studies of Candidatus Thiomargarita nelsonii that suggest metabolic plasticity and mixotrophic metabolism. The cellular oxidoreductase response of bacteria attached to the exterior of Thiomargarita also supports the possibility of trophic interactions between these largest of known bacteria and attached epibionts.IMPORTANCE The metabolic potential of many microorganisms that cannot be grown in the laboratory is known only from genomic data. Genomes of Thiomargarita spp. suggest that these largest of known bacteria are mixotrophs, combining lithotrophic metabolism with organic carbon degradation. Our use of a redox-sensitive tetrazolium dye to query the metabolism of these bacteria provides an independent line of evidence that corroborates the apparent metabolic plasticity of Thiomargarita observed in recently produced genomes. Finding new cultivation-independent means of testing genomic results is critical to testing genome-derived hypotheses on the metabolic potentials of uncultivated microorganisms.

Intercellular transfer along the trichomes of the invasive terminal heterocyst forming cyanobacterium Cylindrospermopsis raciborskii CS-505.

Cylindrospermopsis raciborskii CS-505 is an invasive freshwater filamentous cyanobacterium that when grown diazotrophically may develop trichomes of up to 100 vegetative cells while differentiating only two end heterocysts, the sole sites for their N2-fixation process. We examined the diazotrophic growth and intercellular transfer mechanisms in C. raciborskii CS-505. Subjecting cultures to a combined-nitrogen-free medium to elicit N2 fixation, the trichome length remained unaffected while growth rates decreased. The structures and proteins for intercellular communication showed that while a continuous periplasmic space was apparent along the trichomes, the putative septal junction sepJ gene is divided into two open reading frames and lacks several transmembrane domains unlike the situation in Anabaena, differentiating a 5-fold higher frequency of heterocysts. FRAP analyses also showed that the dyes calcein and 5-CFDA were taken up by heterocysts and vegetative cells, and that the transfer from heterocysts and 'terminal' vegetative cells showed considerably higher transfer rates than that from vegetative cells located in the middle of the trichomes. The data suggest that C. raciborskii CS-505 compensates its low-frequency heterocyst phenotype by a highly efficient transfer of the fixed nitrogen towards cells in distal parts of the trichomes (growing rapidly) while cells in central parts suffers (slow growth).

Metagenome sequencing of the microbial community of a solar saltern crystallizer pond at cáhuil lagoon, chile.

Cáhuil Lagoon in central Chile harbors distinct microbial communities in various solar salterns that are arranged as interconnected ponds with increasing salt concentrations. Here, we report the metagenome of the 3.0- to 0.2-µm fraction of the microbial community present in a crystallizer pond with 34% salinity.

Dinitrogen fixation is restricted to the terminal heterocysts in the invasive cyanobacterium Cylindrospermopsis raciborskii CS-505.

The toxin producing nitrogen-fixing heterocystous freshwater cyanobacterium Cylindrospermopsis raciborskii recently radiated from its endemic tropical environment into sub-tropical and temperate regions, a radiation likely to be favored by its ability to fix dinitrogen (diazotrophy). Although most heterocystous cyanobacteria differentiate regularly spaced intercalary heterocysts along their trichomes when combined nitrogen sources are depleted, C. raciborskii differentiates only two terminal heterocysts (one at each trichome end) that can reach >100 vegetative cells each. Here we investigated whether these terminal heterocysts are the exclusive sites for dinitrogen fixation in C. raciborskii. The highest nitrogenase activity and NifH biosynthesis (western-blot) were restricted to the light phase of a 12/12 light/dark cycle. Separation of heterocysts and vegetative cells (sonication and two-phase aqueous polymer partitioning) demonstrated that the terminal heterocysts are the sole sites for nifH expression (RT-PCR) and NifH biosynthesis. The latter finding was verified by the exclusive localization of nitrogenase in the terminal heterocysts of intact trichomes (immunogold-transmission electron microscopy and in situ immunofluorescence-light microscopy). These results suggest that the terminal heterocysts provide the combined nitrogen required by the often long trichomes (>100 vegetative cells). Our data also suggests that the terminal-heterocyst phenotype in C. raciborskii may be explained by the lack of a patL ortholog. These data help identify mechanisms by which C. raciborskii and other terminal heterocyst-forming cyanobacteria successfully inhabit environments depleted in combined nitrogen.

Co-occurrence of microcystin and microginin congeners in Brazilian strains of Microcystis sp.

Species of Microcystis are the most common bloom-forming cyanobacteria in several countries. Despite extensive studies regarding the production of bioactive cyanopeptides in this genus, there are limited data on isolated strains from Brazil. Three Microcystis sp. strains were isolated from the Salto Grande Reservoir (LTPNA01, 08 and 09) and investigated for the presence of mcy genes, microcystins and other cyanopeptides. Microcystin and microginin production was confirmed in two isolates using high-resolution tandem mass spectrometry after electrospray ionization (ESI-Q-TOF), and the structures of two new microginin congeners were proposed (MG756 Ahda-Val-Leu-Hty-Tyr and MG770 MeAhda-Val-Leu-Hty-Tyr). The biosynthesis profile of the identified cyanopeptides was evaluated at different growth phases via a newly developed HPLC-UV method. Results demonstrated no substantial differences in the production of microcystins and microginins after data normalization to cell quota, suggesting a constitutive biosynthesis. This study represents the first confirmed co-production of microginins and microcystins in Brazilian strains of Microcystis sp. and highlights the potential of Brazilian cyanobacteria as a source of natural compounds with pharmaceutical interest.