Fernando Puente-Sánchez

Horizontal Gene Transfer of Phytochelatin Synthases from Bacteria to Extremophilic Green Algae

Transcriptomic sequencing together with bioinformatic analyses and an automated annotation process led us to identify novel phytochelatin synthase (PCS) genes from two extremophilic green algae (Chlamydomonas acidophila and Dunaliella acidophila). These genes are of intermediate length compared to known PCS genes from eukaryotes and PCS-like genes from prokaryotes. A detailed phylogenetic analysis gives new insight into the complicated evolutionary history of PCS genes and provides evidence for multiple horizontal gene transfer events from bacteria to eukaryotes within the gene family. A separate subgroup containing PCS-like genes within the PCS gene family is not supported since the PCS genes are monophyletic only when the PCS-like genes are included. The presence and functionality of the novel genes in the organisms were verified by genomic sequencing and qRT-PCR. Furthermore, the novel PCS gene in Chlamydomonas acidophila showed very strong induction by cadmium. Cloning and expression of the gene in Escherichia coli clearly improves its cadmium resistance. The gene in Dunaliella was not induced, most likely due to gene duplication.

Comparative Transcriptomic Analysis of the Response of Dunaliella acidophila (Chlorophyta) to Short-Term Cadmium and Chronic Natural Metal-Rich Water Exposures

Heavy metals are toxic compounds known to cause multiple and severe cellular damage. However, acidophilic extremophiles are able to cope with very high concentrations of heavy metals. This study investigated the stress response under natural environmental heavy metal concentrations in an acidophilic Dunaliella acidophila. We employed Illumina sequencing for a de novo transcriptome assembly and to identify changes in response to high cadmium concentrations and natural metal-rich water. The photosynthetic performance was also estimated by pulse amplitude-modulated (PAM) fluorescence. Transcriptomic analysis highlights a number of processes mainly related to a high constitutive expression of genes involved in oxidative stress and response to reactive oxygen species (ROS), even in the absence of heavy metals. Photosynthetic activity seems to be unaltered under short-term exposition to Cd and chronic exposure to natural metal-rich water, probably due to an increase in the synthesis of structural photosynthetic components preserving their functional integrity. An overrepresentation of Gene Ontology (GO) terms related to metabolic activities, transcription, and proteosomal catabolic process was observed when D. acidophila grew under chronic exposure to natural metal-rich water. GO terms involved in carbohydrate metabolic process, reticulum endoplasmic and Golgi bodies, were also specifically overrepresented in natural metal-rich water library suggesting an endoplasmic reticulum stress response.

Nucleation of Fe-rich phosphates and carbonates on microbial cells and exopolymeric substances

Although phosphate and carbonate are important constituents in ancient and modern environments, it is not yet clear their biogeochemical relationships and their mechanisms of formation. Microbially mediated carbonate formation has been widely studied whereas little is known about the formation of phosphate minerals. Here we report that a new bacterial strain, Tessarococcus lapidicaptus, isolated from the subsurface of Rio Tinto basin (Huelva, SW Spain), is capable of precipitating Fe-rich phosphate and carbonate minerals. We observed morphological differences between phosphate and carbonate, which may help us to recognize these minerals in terrestrial and extraterrestrial environments. Finally, considering the scarcity and the unequal distribution and preservation patterns of phosphate and carbonates, respectively, in the geological record and the biomineralization process that produces those minerals, we propose a hypothesis for the lack of Fe-phosphates in natural environments and ancient rocks.

Solar Radiation Stress in Natural Acidophilic Biofilms of Euglena mutabilis Revealed by Metatranscriptomics and PAM Fluorometry.

The daily photosynthetic performance of a natural biofilm of the extreme acidophilic Euglena mutabilis from Río Tinto (SW, Spain) under full solar radiation was analyzed by means of pulse amplitude-modulated (PAM) fluorescence measurements and metatrascriptomic analysis. Natural E. mutabilis biofilms undergo large-scale transcriptomic reprogramming during midday due to a dynamic photoinhibition and solar radiation stress. Photoinhibition is due to UV radiation and not to light intensity, as revealed by PAM fluorometry analysis. In order to minimize the negative effects of solar radiation, our data supports the presence of a circadian rhythm in this euglenophyte that increases their opportunity to survive. Differential gene expression throughout the day (at 12:00, 20:00 and night) was monitored by massive Illumina parallel sequencing of metatranscriptomic libraries. The transcription pattern was altered in genes involved in Photosystem II stability and repair, UV damaged DNA repair, non-photochemical quenching and oxidative stress, supporting the photoinhibition detected by PAM fluorometry at midday.

SqueezeM, a fully automatic metagenomic analysis pipeline from reads to bins

SqueezeM is a full automatic pipeline for metagenomics/metatranscriptomics, covering all steps of the analysis. In contrast to other available tools, SqueezeM includes multi-metagenome support allowing the co-assembly of related metagenomes and the retrieval of individual genomes via binning procedures. Thus, Squeeze M features several unique characteristics: Coassembly procedure with read mapping for estimation of the abundances of genes in each metagenome, or coassembly of unlimited number of metagenomes via merging of individual metagenomes. It also includes binning and bin checking, for retrieving individual genomes. Internal checks for the assembly and binning steps inform about the consistency of contigs and bins, allowing to spot potential chimeras. Metatranscriptomic support via mapping of cDNA reads against reference metagenomes. The results are stored in a database, where they can be easily exported and shared, and can be inspected anywhere using a web interface.The improvement of sequencing technologies has allowed the generalization of metagenomic sequencing, which has become a standard procedure for analysing the structure and functionality of microbiomes. The bioinformatic analysis of the sequencing results poses a challenge because it involves many different complex steps. SqueezeMeta is a full automatic pipeline for metagenomics/metatranscriptomics, covering all steps of the analysis. SqueezeMeta includes multi-metagenome support allowing the co-assembly of related metagenomes and the retrieval of individual genomes via binning procedures. SqueezeMeta features several unique characteristics: Co-assembly procedure or co-assembly of unlimited number of metagenomes via merging of individual assembled metagenomes, both with read mapping for estimation of the abundances of genes in each metagenome. It also includes binning and bin checking for retrieving individual genomes. Internal checks for the assembly and binning steps inform about the consistency of contigs and bins. Also, the results are stored in a mySQL database, where they can be easily exported and shared, and can be inspected anywhere using a flexible web interface allowing the easy creation of complex queries. We illustrate the potential of SqueezeMeta by analyzing 32 gut metagenomes in a fully automatic way, allowing to retrieve several millions of genes and several hundreds of genomic bins. One of the motivations in the development of SqueezeMeta was producing a software capable to run in small desktop computers, thus being amenable to all users and all settings. We were also able to co-assemble two of these metagenomes and complete the full analysis in less than one day using a simple laptop computer, illustrating the capacity of SqueezeMeta to run without high-performance computing infrastructure. SqueezeMeta is a complete system covering all steps in the analysis of metagenomes and metatranscriptomes, capable to work even in scarcity of computational resources. It is therefore adequate for in-situ, real time analysis of metagenomes produced by nanopore sequencing. SqueezeMeta can be downloaded from https://github.com/jtamames/SqueezeMeta

Basis of genetic adaptation to heavy metal stress in the acidophilic green alga Chlamydomonas acidophila

To better understand heavy metal tolerance in Chlamydomonas acidophila, an extremophilic green alga, we assembled its transcriptome and measured transcriptomic expression before and after Cd exposure in this and the neutrophilic model microalga Chlamydomonas reinhardtii. Genes possibly related to heavy metal tolerance and detoxification were identified and analyzed as potential key innovations that enable this species to live in an extremely acid habitat with high levels of heavy metals. In addition we provide a data set of single orthologous genes from eight green algal species as a valuable resource for comparative studies including eukaryotic extremophiles. Our results based on differential gene expression, detection of unique genes and analyses of codon usage all indicate that there are important genetic differences in C. acidophila compared to C. reinhardtii. Several efflux family proteins were identified as candidate key genes for adaptation to acid environments. This study suggests for the first time that exposure to cadmium strongly increases transposon expression in green algae, and that oil biosynthesis genes are induced in Chlamydomonas under heavy metal stress. Finally, the comparison of the transcriptomes of several acidophilic and non-acidophilic algae showed that the Chlamydomonas genus is polyphyletic and that acidophilic algae have distinctive aminoacid usage patterns.

Environmental parameters, and not phylogeny, determine the composition of extracellular polymeric substances in microbial mats from extreme environments

The ability to establish biofilms is a key trait for microorganisms growing in extreme environments. The extracellular polymeric substances (EPS) present in biofilms provide not only surface attachment, but also protection against all kinds of environmental stressors, including desiccation, salinity, temperature or heavy metal pollution. The acquisition of suitable biofilm characteristics might thus be an important process mediating the adaptation of microorganisms to novel environmental conditions. In this work we have characterized the EPS of 20 phylogenetically diverse biofilms collected in situ from five contrasting extreme environments, including two geothermal areas (Copahue, Argentina; Seltun, Iceland), two cold areas (Pastoruri glacier, Peru; Byers Peninsula, Antarctica) and one extremely acidic river (Río Tinto, Spain). Biofilms were subjected to biochemical characterization, glycan profiling and immunoprofiling with an antibody microarray. Our results showed that environmental conditions strongly influence biofilm characteristics, with microorganisms from the same environment achieving similar EPS compositions regardless of the phylogeny of their main species. The concentration of some monosaccharides in the EPS could be related to environmental conditions such as temperature or heavy metal toxicity, suggesting that in some cases stress resistance can be mediated by specific sugars. Overall, our results highlight the existence of conserved EPS compositional patterns for each extreme environment, which could in turn be exploited to engineer ecological adaptations in genetically modified microorganisms.

Draft Genome Sequence of the Deep-Subsurface Actinobacterium Tessaracoccus lapidicaptus IPBSL-7T.

ABSTRACT The type strain of Tessaracoccus lapidicaptus was isolated from the deep subsurface of the Iberian Pyrite Belt (southwest Spain). Here, we report its draft genome, consisting of 27 contigs with a ~3.1-Mb genome size. The annotation revealed 2,905 coding DNA sequences, 45 tRNA genes, and three rRNA genes.