Maximizing content across scales: Moving multimodal microscopy and mesoscopy toward molecular imaging.

Molecular imaging aims to depict the molecules in living patients. However, because this aim is still far beyond reach, patchworks of different solutions need to be used to tackle this overarching goal. From the vast toolbox of imaging techniques, we focus on those recent advances in optical microscopy that image molecules and cells at the submicron to centimeter scale. Mesoscopic imaging covers the "imaging gap" between techniques such as confocal microscopy and magnetic resonance imagingthat image entire live samples but with limited resolution. Microscopy focuses on the cellular level; mesoscopy visualizes the organization of molecules and cells into tissues and organs. The correlation between these techniques allows us to combine disciplines ranging from whole body imaging to basic research of model systems. We review current developments focused on improving microscopic and mesoscopic imaging technologies and on hardware and software that push the current sensitivity and resolution boundaries.

Winter diversity and expression of proteorhodopsin genes in a polar ocean.

Mixotrophy is a valuable functional trait used by microbes when environmental conditions vary broadly or resources are limited. In the sunlit waters of the ocean, photoheterotrophy, a form of mixotrophy, is often mediated by proteorhodopsin (PR), a seven helices transmembrane protein binding the retinal chromophore. Altogether, they allow bacteria to capture photic energy for sensory and proton gradient formation cell functions. The seasonal occurrence and diversity of the gene coding for PR in cold oligotrophic polar oceans is not known and PR expression has not yet been reported. Here we show that PR is widely distributed among bacterial taxa, and that PR expression decreased markedly during the winter months in the Arctic Ocean. Gammaproteobacteria-like PR sequences were always dominant. However, within the second most common affiliation, there was a transition from Flavobacteria-like PR in early winter to Alphaproteobacteria-like PR in late winter. The phylogenetic shifts followed carbon dynamics, where patterns in expression were consistent with community succession, as identified by DNA community fingerprinting. Although genes for PR were always present, the trend in decreasing transcripts from January to February suggested reduced functional utility of PR during winter. Under winter darkness, sustained expression suggests that PR may continue to be useful for non-ATP forming functions, such as environmental sensing or small solute transport. The persistence of PR expression in winter among some bacterial groups may offer a competitive advantage, where its multifunctionality enhances microbial survival under harsh polar conditions.

Genomics of the proteorhodopsin-containing marine flavobacterium Dokdonia sp. strain MED134.

Proteorhodopsin phototrophy is expected to have considerable impact on the ecology and biogeochemical roles of marine bacteria. However, the genetic features contributing to the success of proteorhodopsin-containing bacteria remain largely unknown. We investigated the genome of Dokdonia sp. strain MED134 (Bacteroidetes) for features potentially explaining its ability to grow better in light than darkness. MED134 has a relatively high number of peptidases, suggesting that amino acids are the main carbon and nitrogen sources. In addition, MED134 shares with other environmental genomes a reduction in gene copies at the expense of important ones, like membrane transporters, which might be compensated by the presence of the proteorhodopsin gene. The genome analyses suggest Dokdonia sp. MED134 is able to respond to light at least partly due to the presence of a strong flavobacterial consensus promoter sequence for the proteorhodopsin gene. Moreover, Dokdonia sp. MED134 has a complete set of anaplerotic enzymes likely to play a role in the adaptation of the carbon anabolism to the different sources of energy it can use, including light or various organic matter compounds. In addition to promoting growth, proteorhodopsin phototrophy could provide energy for the degradation of complex or recalcitrant organic matter, survival during periods of low nutrients, or uptake of amino acids and peptides at low concentrations. Our analysis suggests that the ability to harness light potentially makes MED134 less dependent on the amount and quality of organic matter or other nutrients. The genomic features reported here may well be among the keys to a successful photoheterotrophic lifestyle.

Transcriptome fingerprinting analysis: an approach to explore gene expression patterns in marine microbial communities.

Microbial transcriptomics are providing new insights into the functional processes of microbial communities. However, analysis of each sample is still expensive and time consuming. A rapid and low cost method that would allow the identification of the most interesting samples for posterior in-depth metatranscriptomics analysis would be extremely useful. Here we present Transcriptome Fingerprinting Analysis (TFA) as an approach to fulfill this objective in microbial ecology studies. We have adapted the differential display technique for mRNA fingerprinting based on the PCR amplification of expressed transcripts to interrogate natural microbial eukaryotic communities. Unlike other techniques, TFA does not require prior knowledge of the mRNA sequences to be detected. We have used a set of arbitrary primers coupled with a fluorescence labeled primer targeting the poly(A) tail of the eukaryotic mRNA, with further detection of the resulting labeled cDNA products in an automated genetic analyzer. The output represented by electropherogram peak patterns allowed the comparison of a set of genes expressed at the time of sampling. TFA has been optimized by testing the sensitivity of the method for different initial RNA amounts, and the repeatability of the gene expression patterns with increasing time after sampling both with cultures and environmental samples. Results show that TFA is a promising approach to explore the dynamics of gene expression patterns in microbial communities.

Genome analysis of the proteorhodopsin-containing marine bacterium Polaribacter sp. MED152 (Flavobacteria).

Analysis of marine cyanobacteria and proteobacteria genomes has provided a profound understanding of the life strategies of these organisms and their ecotype differentiation and metabolisms. However, a comparable analysis of the Bacteroidetes, the third major bacterioplankton group, is still lacking. In the present paper, we report on the genome of Polaribacter sp. strain MED152. On the one hand, MED152 contains a substantial number of genes for attachment to surfaces or particles, gliding motility, and polymer degradation. This agrees with the currently assumed life strategy of marine Bacteroidetes. On the other hand, it contains the proteorhodopsin gene, together with a remarkable suite of genes to sense and respond to light, which may provide a survival advantage in the nutrient-poor sun-lit ocean surface when in search of fresh particles to colonize. Furthermore, an increase in CO(2) fixation in the light suggests that the limited central metabolism is complemented by anaplerotic inorganic carbon fixation. This is mediated by a unique combination of membrane transporters and carboxylases. This suggests a dual life strategy that, if confirmed experimentally, would be notably different from what is known of the two other main bacterial groups (the autotrophic cyanobacteria and the heterotrophic proteobacteria) in the surface oceans. The Polaribacter genome provides insights into the physiological capabilities of proteorhodopsin-containing bacteria. The genome will serve as a model to study the cellular and molecular processes in bacteria that express proteorhodopsin, their adaptation to the oceanic environment, and their role in carbon-cycling.

Light stimulates growth of proteorhodopsin-containing marine Flavobacteria.

Proteorhodopsins are bacterial light-dependent proton pumps. Their discovery within genomic material from uncultivated marine bacterioplankton caused considerable excitement because it indicated a potential phototrophic function within these organisms, which had previously been considered strictly chemotrophic. Subsequent studies established that sequences encoding proteorhodopsin are broadly distributed throughout the world's oceans. Nevertheless, the role of proteorhodopsins in native marine bacteria is still unknown. Here we show, from an analysis of the complete genomes of three marine Flavobacteria, that cultivated bacteria in the phylum Bacteroidetes, one of the principal components of marine bacterioplankton, contain proteorhodopsin. Moreover, growth experiments in both natural and artificial seawater (low in labile organic matter, which is typical of the world's oceans) establish that exposure to light results in a marked increase in the cell yield of one such bacterium (Dokdonia sp. strain MED134) when compared with cells grown in darkness. Thus, our results show that the phototrophy conferred by proteorhodopsin can provide critical amounts of energy, not only for respiration and maintenance but also for active growth of marine bacterioplankton in their natural environment.