Expressió gènica en microorganismes marins

Ara que es comença a treure l’entrellat -o si més no a tenir una nova perspectiva- de la diversitat de microorganismes present als oceans gràcies a la biologia molecular i a la metagenòmica, el següent pas és esbrinar quines són les noves funcions que aquesta amaga i com són utilitzades i per qui a l’oceà. La regulació de l’expressió gènica és la base de la versatilitat i l’adaptabilitat de qualsevol organisme al medi on viu. De l’estudi dels gens expressats d’un organisme es poden deduir correctament moltes de les característiques del medi on la seva vida es desenvolupa habitualment. En cada situació els organismes expressen solament una part del seus gens, responent tant a factors interns (per exemple el cicle cel·lular) com a factors externs (temperatura, llum, aport de nutrients...). Les tecnologies de seqüenciació massiva també s’han aplicat en l’estudi de l’expressió de les comunitats microbianes marines (metatranscriptòmica). Tanmateix, aquestes tecnologies encara no estan prou optimitzades i sovint proporcionen seqüències que no poden ser assignades a cap gen conegut. En aquesta tesi ens hem plantejat l’estudi de l’expressió gènica dels microorganismes marins a tres escales diferents: a escala de comunitat, de genoma, i de gen. L’esforç més gran ha estat estudiar l’expressió gènica a escala de comunitat, on el nostre repte ha estat desenvolupar una tècnica equivalent als mètodes “d’empremta dactilar” (fingerprinting) del DNA que s’usen de forma rutinària –com la DGGE o l’ARISA- per tal d’explorar la dinàmica dels patrons d’expressió gènica de les comunitats de microbis marins, permetent la comparació d’un gran nombre de mostres a un preu assequible i sense la necessitat prèvia de saber les seqüències dels RNA missatgers. Aquesta tècnica, batejada com a TFA (de “Transcriptome Fingerprinting Analysis”), ens ha permès estudiar I) les variacions estacionals en els patrons d’expressió gènica dels picoeucariotes marins de l’Observatori Microbià de la Badia de Blanes durant 4 anys, i II) les variacions dels patrons d’expressió al llarg d’un gradient espacial horitzontal i vertical i d’un gradient temporal. En ambdós casos, els canvis d’expressió s’han comparat amb els canvis en l’estructura de la comunitat (mitjançant l’ARISA). A escala genòmica hem estudiat la resposta transcripcional global d’un microorganisme heteròtrof a la llum. La llum és responsable d’una gran quantitat de respostes fisiològiques. Una gran part dels microorganismes del mar que utilitzen la llum ho fan mitjançant la fotosíntesis, però existeixen d’altres microorganismes que utilitzen la llum de manera diferent, com els fotoheteròtrofs, que la utilitzen per generar energia però no fixen CO2. En un dels estudis de genòmica ambiental es va descobrir la presència d’una proteïna fotoactiva, la proteorodopsina, associada a un grup de bacteris marins no cultivats. Les proteorodopsines són responsables d’un nou mecanisme de fototrofia als oceans; funcionen com a bombes de protons accionades per la llum que generen un gradient de protons a la membrana per tal de sintetitzar ATP. A escala de gen, en aquesta tesi hem estudiat mitjançant RT-PCR l’expressió del gen de la proteorodopsina en un cultiu d’una flavobacteria marina i hem vist que la llum augmentava els seus nivells d’expressió.Recent advances have been crucial to understand, or at least to have a new perspective, on the diversity of microorganisms present in the oceans through molecular biology and metagenomics. The next step is to find out what functions are hidden within this diversity and how and when are they used. The regulation of gene expression is the basis of the versatility and adaptability of any living organism to the environment. The study of the genes expressed in an organism can help to deduce many of the characteristics of the environment. Usually, organisms express only a portion of their genes in response to both internal factors (e.g. cell cycle) and external factors (temperature, light, nutrients, etc.). Massive sequencing technologies have also been applied to the study of the expression of genes in marine microbial communities (metatranscriptomics). However, these technologies are not yet sufficiently optimized and often provide sequences that cannot be assigned to known genes. In this PhD thesis I have studied gene expression of marine organisms at three different levels: at the community level, at the genome level, and at the gene level. The major effort was dedicated to gene expression at the community level, where the challenge was to develop a technique equivalent to DNA fingerprinting methods that are routinely used -such as ARISA or DGGE- in order to explore the dynamics of gene expression patterns in marine microbial communities, allowing the comparison of a large number of samples at an affordable price and without the need for prior knowledge of the messenger RNA sequences. This technique, called TFA (from “Transcriptome Fingerprinting Analysis”), has then been used to study I) seasonal variations in gene expression patterns of marine picoeukaryotes at the Blanes Bay Microbial Observatory during 4 years, and II) changes in expression patterns along spatial horizontal and vertical gradients and diel cycles. In both cases, expression changes were compared with changes in community structure (by ARISA). At the genomic level I have studied the global transcriptional response to light of a heterotrophic microorganism. Light is responsible for a large number of physiological responses. A large fraction of marine microorganisms that use light perform photosynthesis, but there are other organisms as photo-heterotrophs, who use light to generate energy but do not fix CO2. At the gene level, we have studied the proteorhodopsin gene expression by RT-PCR in a culture of a marine flavobacterium. In a study of environmental genomics, the presence of this photoactive protein was found to be associated with a group of uncultivated marine bacteria. Proteorhodopsins are responsible of a new mechanism of phototrophy in the oceans; they act as proton pumps powered by light that generate a membrane proton gradient in order to synthesize ATP. In the present study it was found that light increased the expression levels of the proteorhodopsin gene.Postprint (published version

Expressió gènica en microorganismes marins

Ara que es comença a treure l’entrellat -o si més no a tenir una nova perspectiva- de la diversitat de microorganismes present als oceans gràcies a la biologia molecular i a la metagenòmica, el següent pas és esbrinar quines són les noves funcions que aquesta amaga i com són utilitzades i per qui a l’oceà. La regulació de l’expressió gènica és la base de la versatilitat i l’adaptabilitat de qualsevol organisme al medi on viu. De l’estudi dels gens expressats d’un organisme es poden deduir correctament moltes de les característiques del medi on la seva vida es desenvolupa habitualment. En cada situació els organismes expressen solament una part del seus gens, responent tant a factors interns (per exemple el cicle cel·lular) com a factors externs (temperatura, llum, aport de nutrients...). Les tecnologies de seqüenciació massiva també s’han aplicat en l’estudi de l’expressió de les comunitats microbianes marines (metatranscriptòmica). Tanmateix, aquestes tecnologies encara no estan prou optimitzades i sovint proporcionen seqüències que no poden ser assignades a cap gen conegut. En aquesta tesi ens hem plantejat l’estudi de l’expressió gènica dels microorganismes marins a tres escales diferents: a escala de comunitat, de genoma, i de gen. L’esforç més gran ha estat estudiar l’expressió gènica a escala de comunitat, on el nostre repte ha estat desenvolupar una tècnica equivalent als mètodes “d’empremta dactilar” (fingerprinting) del DNA que s’usen de forma rutinària –com la DGGE o l’ARISA- per tal d’explorar la dinàmica dels patrons d’expressió gènica de les comunitats de microbis marins, permetent la comparació d’un gran nombre de mostres a un preu assequible i sense la necessitat prèvia de saber les seqüències dels RNA missatgers. Aquesta tècnica, batejada com a TFA (de “Transcriptome Fingerprinting Analysis”), ens ha permès estudiar I) les variacions estacionals en els patrons d’expressió gènica dels picoeucariotes marins de l’Observatori Microbià de la Badia de Blanes durant 4 anys, i II) les variacions dels patrons d’expressió al llarg d’un gradient espacial horitzontal i vertical i d’un gradient temporal. En ambdós casos, els canvis d’expressió s’han comparat amb els canvis en l’estructura de la comunitat (mitjançant l’ARISA). A escala genòmica hem estudiat la resposta transcripcional global d’un microorganisme heteròtrof a la llum. La llum és responsable d’una gran quantitat de respostes fisiològiques. Una gran part dels microorganismes del mar que utilitzen la llum ho fan mitjançant la fotosíntesis, però existeixen d’altres microorganismes que utilitzen la llum de manera diferent, com els fotoheteròtrofs, que la utilitzen per generar energia però no fixen CO2. En un dels estudis de genòmica ambiental es va descobrir la presència d’una proteïna fotoactiva, la proteorodopsina, associada a un grup de bacteris marins no cultivats. Les proteorodopsines són responsables d’un nou mecanisme de fototrofia als oceans; funcionen com a bombes de protons accionades per la llum que generen un gradient de protons a la membrana per tal de sintetitzar ATP. A escala de gen, en aquesta tesi hem estudiat mitjançant RT-PCR l’expressió del gen de la proteorodopsina en un cultiu d’una flavobacteria marina i hem vist que la llum augmentava els seus nivells d’expressió.Recent advances have been crucial to understand, or at least to have a new perspective, on the diversity of microorganisms present in the oceans through molecular biology and metagenomics. The next step is to find out what functions are hidden within this diversity and how and when are they used. The regulation of gene expression is the basis of the versatility and adaptability of any living organism to the environment. The study of the genes expressed in an organism can help to deduce many of the characteristics of the environment. Usually, organisms express only a portion of their genes in response to both internal factors (e.g. cell cycle) and external factors (temperature, light, nutrients, etc.). Massive sequencing technologies have also been applied to the study of the expression of genes in marine microbial communities (metatranscriptomics). However, these technologies are not yet sufficiently optimized and often provide sequences that cannot be assigned to known genes. In this PhD thesis I have studied gene expression of marine organisms at three different levels: at the community level, at the genome level, and at the gene level. The major effort was dedicated to gene expression at the community level, where the challenge was to develop a technique equivalent to DNA fingerprinting methods that are routinely used -such as ARISA or DGGE- in order to explore the dynamics of gene expression patterns in marine microbial communities, allowing the comparison of a large number of samples at an affordable price and without the need for prior knowledge of the messenger RNA sequences. This technique, called TFA (from “Transcriptome Fingerprinting Analysis”), has then been used to study I) seasonal variations in gene expression patterns of marine picoeukaryotes at the Blanes Bay Microbial Observatory during 4 years, and II) changes in expression patterns along spatial horizontal and vertical gradients and diel cycles. In both cases, expression changes were compared with changes in community structure (by ARISA). At the genomic level I have studied the global transcriptional response to light of a heterotrophic microorganism. Light is responsible for a large number of physiological responses. A large fraction of marine microorganisms that use light perform photosynthesis, but there are other organisms as photo-heterotrophs, who use light to generate energy but do not fix CO2. At the gene level, we have studied the proteorhodopsin gene expression by RT-PCR in a culture of a marine flavobacterium. In a study of environmental genomics, the presence of this photoactive protein was found to be associated with a group of uncultivated marine bacteria. Proteorhodopsins are responsible of a new mechanism of phototrophy in the oceans; they act as proton pumps powered by light that generate a membrane proton gradient in order to synthesize ATP. In the present study it was found that light increased the expression levels of the proteorhodopsin gene

Genomics of the Proteorhodopsin-Containing Marine Flavobacterium Dokdonia sp. Strain MED134

González, José M. ... et al.-- 12 pages, 8 figures, 1 table, supplemental material http://aem.asm.org/content/77/24/8676/suppl/DC1Proteorhodopsin 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 lifestyleJ.M.G. and C.P.-A. were supported by grant CTM2010-11060-E from the Spanish Ministry of Science and Innovation, J.P. was supported by the Swedish Research Council and FORMAS, and P.P. was supported by the intramural funds of the U.S. Department of Health and Human Services (National Library of Medicine, National Institutes of Health)Peer reviewe

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

Are octopus really aliens? On the fine anatomical and histological details of one of the most intriguing sea creatures

19th International European Light Microscopy Initiative Meeting, 4-7 June 2019.-- 1 page, figure

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 communitiesMC-L was the recipient of an FPI fellowship from the Spanish Ministry of Science and Innovation. This work was funded by grant GEMMA (CTM2007- 63753-C02-01/MAR) from the Spanish Ministry of Science and Innovation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Transitional characters to the benthic lifestyle in juvenile merobenthic octopuses

Cephalopod International Advisory Council Conference (CIAC 2018) : Cephalopod Research Across Scales: From Molecules to Ecosystems, 12-16 November 2018, St. Petersburg, Florida, USA.-- 1 pageMerobenthic octopuses produce numerous small eggs that hatch into planktonic, free-swimming hatchlings with few suckers, simple chromatophores and transparent musculature. After a planktonic period that can range from few weeks to half a year, depending on the species and temperature, a major metamorphosis occurs in morphology, physiology and behaviour when animals settle on the sea bottom. At settlement, a positive allometric arm growth emerges and their body surface generates chromatophores, iridiophores and leucophores, and skin sculptural components develop. At the same time, they seems to lose the Kölliker organs, whose function is unknown, as well as the lateral line system. These organs are present on its external epithelium from hatching, however, they have never been reported from the skin of subadult and adult benthic octopods. These structures and their possible presence, transformation and/or degeneration are carefully searched here over the skin body surface of recently settled juveniles as well as in subadult merobenthic octopuses Eledone cirrhosa and Octopus vulgaris. Techniques used are Scanning Electron Microscopy (SEM) and Selective Plane Illumination Microscopy (SPIM). All of these morphological changes are discussed in the context of the ontogenetic transition towards a benthic lifestylePeer Reviewe

Scheme of the different steps in Transcriptome Fingerprinting Analysis (TFA).

<p>Note that in step 4 there is a mixture of ribosomal, transfer, and messenger RNAs. By using primers against the poly(A) tail, step 5 reverse-transcribe mRNAs only.</p

Examples of TFA profiles, showing the sensitivity and repeatability of the technique.

<p>All panels are replicates of the same environmental sample. Panels A and B show replicate fingerprints obtained from 20 ng of total RNA and panels C and D from 40 ng of RNA. The horizontal scale goes from 330 to 390 bp from left to right.</p

Location and depths of samples analyzed in Figure 6.

<p>Location and depths of samples analyzed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022950#pone-0022950-g006" target="_blank">Figure 6</a>.</p