Macroalgal microbiomes unveil a valuable genetic resource for halogen metabolism.

BACKGROUND: Macroalgae, especially reds (Rhodophyta Division) and browns (Phaeophyta Division), are known for producing various halogenated compounds. Yet, the reasons underlying their production and the fate of these metabolites remain largely unknown. Some theories suggest their potential antimicrobial activity and involvement in interactions between macroalgae and prokaryotes. However, detailed investigations are currently missing on how the genetic information of prokaryotic communities associated with macroalgae may influence the fate of organohalogenated molecules. RESULTS: To address this challenge, we created a specialized dataset containing 161 enzymes, each with a complete enzyme commission number, known to be involved in halogen metabolism. This dataset served as a reference to annotate the corresponding genes encoded in both the metagenomic contigs and 98 metagenome-assembled genomes (MAGs) obtained from the microbiome of 2 red (Sphaerococcus coronopifolius and Asparagopsis taxiformis) and 1 brown (Halopteris scoparia) macroalgae. We detected many dehalogenation-related genes, particularly those with hydrolytic functions, suggesting their potential involvement in the degradation of a wide spectrum of halocarbons and haloaromatic molecules, including anthropogenic compounds. We uncovered an array of degradative gene functions within MAGs, spanning various bacterial orders such as Rhodobacterales, Rhizobiales, Caulobacterales, Geminicoccales, Sphingomonadales, Granulosicoccales, Microtrichales, and Pseudomonadales. Less abundant than degradative functions, we also uncovered genes associated with the biosynthesis of halogenated antimicrobial compounds and metabolites. CONCLUSION: The functional data provided here contribute to understanding the still largely unexplored role of unknown prokaryotes. These findings support the hypothesis that macroalgae function as holobionts, where the metabolism of halogenated compounds might play a role in symbiogenesis and act as a possible defense mechanism against environmental chemical stressors. Furthermore, bacterial groups, previously never connected with organohalogen metabolism, e.g., Caulobacterales, Geminicoccales, Granulosicoccales, and Microtrichales, functionally characterized through MAGs reconstruction, revealed a biotechnologically relevant gene content, useful in synthetic biology, and bioprospecting applications. Video Abstract.

Comparative Genomics Suggests a Taxonomic Revision of the Staphylococcus cohnii Species Complex.

Staphylococcus cohnii (SC), a coagulase-negative bacterium, was first isolated in 1975 from human skin. Early phenotypic analyses led to the delineation of two subspecies (subsp.), Staphylococcus cohnii subsp. cohnii (SCC) and Staphylococcus cohnii subsp. urealyticus (SCU). SCC was considered to be specific to humans, whereas SCU apparently demonstrated a wider host range, from lower primates to humans. The type strains ATCC 29974 and ATCC 49330 have been designated for SCC and SCU, respectively. Comparative analysis of 66 complete genome sequences-including a novel SC isolate-revealed unexpected patterns within the SC complex, both in terms of genomic sequence identity and gene content, highlighting the presence of 3 phylogenetically distinct groups. Based on our observations, and on the current guidelines for taxonomic classification for bacterial species, we propose a revision of the SC species complex. We suggest that SCC and SCU should be regarded as two distinct species: SC and SU (Staphylococcus urealyticus), and that two distinct subspecies, SCC and SCB (SC subsp. barensis, represented by the novel strain isolated in Bari) should be recognized within SC. Furthermore, since large-scale comparative genomics studies recurrently suggest inconsistencies or conflicts in taxonomic assignments of bacterial species, we believe that the approach proposed here might be considered for more general application.

Staphylococcus arlettae Genomics: Novel Insights on Candidate Antibiotic Resistance and Virulence Genes in an Emerging Opportunistic Pathogen.

Coagulase Negative Staphylococci (CoNS) are becoming increasingly recognized as an important cause of human and animal infections. Notwithstanding their clinical relevance, annotation of genes potentially involved in pathogenicity and/or antibiotic resistance in the CoNS species Staphylococcus arlettae (SAR) is currently very limited. In the current work we describe the genome of a novel methicillin resistant isolate of SAR, which we named Bari, and present a comprehensive analysis of predicted antibiotic resistance profiles and virulence determinants for all the 22 currently available SAR genomes. By comparing predicted antibiotic resistance and virulence-associated genes with those obtained from a manual selection of 148 bacterial strains belonging to 14 different species of staphylococci and to two "outgroup" species, Bacillus subtilis (BS) and Macrococcus caseoliticus (MC), we derived some interesting observations concerning the types and number of antibiotic resistance-related and virulence-like genes in SAR. Interestingly, almost 50% of the putative antibiotic resistance determinants identified in this work, which include the clinically relevant mec, van, and cls genes, were shared among all the SAR strains herein considered (Bari included). Moreover, comparison of predicted antibiotic resistance profiles suggest that SAR is closely related to well-known pathogenic Staphylococcus species, such as Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE). A similar analysis of predicted virulence factors, revealed that several genes associated with pathogenesis (including, for example, ica, nuc, and ssp), which are commonly found in the genomes of pathogenic staphylococci such as Staphylococcus haemolyticus (SH) and Staphylococcus saprophyticus (SS), are observed also in the SAR strains for which a genomic sequence is available. All in all, we believe that the analyses presented in the current study, by providing a consistent and comprehensive annotation of virulence and antibiotic resistance-related genes in SAR, can constitute a valuable resource for the study of molecular mechanisms of opportunistic pathogenicity in this species.

Draft Genome Sequences of Three Novel Staphylococcus arlettae Strains Isolated from a Disused Biological Safety Cabinet.

The genome sequences of three new strains of Staphylococcus arlettae named Bari1, Bari2, and Bari3 are presented. The strains exhibited tolerance to hexavalent chromium ions. An sprC gene encoding a putative chromium transporter was present in each of the three draft genome sequences.

A-GAME: improving the assembly of pooled functional metagenomics sequence data.

BACKGROUND: Expression screening of environmental DNA (eDNA) libraries is a popular approach for the identification and characterization of novel microbial enzymes with promising biotechnological properties. In such "functional metagenomics" experiments, inserts, selected on the basis of activity assays, are sequenced with high throughput sequencing technologies. Assembly is followed by gene prediction, annotation and identification of candidate genes that are subsequently evaluated for biotechnological applications. RESULTS: Here we present A-GAME (A GAlaxy suite for functional MEtagenomics), a web service incorporating state of the art tools and workflows for the analysis of eDNA sequence data. We illustrate the potential of A-GAME workflows using real functional metagenomics data, showing that they outperform alternative metagenomics assemblers. Dedicated tools available in A-GAME allow efficient analysis of pooled libraries and rapid identification of candidate genes, reducing sequencing costs and saving the need for laborious manual annotation. CONCLUSION: In conclusion, we believe A-GAME will constitute a valuable resource for the functional metagenomics community. A-GAME is publicly available at http://beaconlab.it/agame.

Cryptochrome 2 extensively regulates transcription of the chloroplast genome in tomato.

Light plays a key role in the regulation of many physiological processes required for plant and chloroplast development. Plant cryptochromes (crys) play an important role in monitoring, capturing, and transmitting the light stimuli. In this study, we analyzed the effects of CRY2 overexpression on transcription of tomato chloroplast genome by a tiling array, containing about 90 000 overlapping probes (5-nucleotide resolution). We profiled transcription in leaves of wild-type and CRY2-overexpressing plants grown in a diurnal cycle, to generate a comprehensive map of chloroplast transcription and to monitor potential specific modulations of the chloroplast transcriptome induced by the overexpression of CRY2. Our results demonstrate that CRY2 is a master gene of transcriptional regulation in the tomato chloroplast. In fact, it modulates the day/night mRNA abundance of about 58% of the 114 ORFs. The effect of CRY2 includes a differential extension of some transcripts at their 5'-end, according to the period of the day. We observed that the influence of CRY2 on chloroplast transcription is not limited to coding RNA; a great number of putative noncoding micro RNA also showed differential accumulation pattern. To our knowledge, this is the first study that highlights how a photoreceptor affects the day/night transcription of the chloroplast genome.

Diversity of hydrolases from hydrothermal vent sediments of the Levante Bay, Vulcano Island (Aeolian archipelago) identified by activity-based metagenomics and biochemical characterization of new esterases and an arabinopyranosidase.

A metagenomic fosmid expression library established from environmental DNA (eDNA) from the shallow hot vent sediment sample collected from the Levante Bay, Vulcano Island (Aeolian archipelago) was established in Escherichia coli. Using activity-based screening assays, we have assessed 9600 fosmid clones corresponding to approximately 350 Mbp of the cloned eDNA, for the lipases/esterases/lactamases, haloalkane and haloacid dehalogenases, and glycoside hydrolases. Thirty-four positive fosmid clones were selected from the total of 120 positive hits and sequenced to yield ca. 1360 kbp of high-quality assemblies. Fosmid inserts were attributed to the members of ten bacterial phyla, including Proteobacteria, Bacteroidetes, Acidobateria, Firmicutes, Verrucomicrobia, Chloroflexi, Spirochaetes, Thermotogae, Armatimonadetes, and Planctomycetes. Of ca. 200 proteins with high biotechnological potential identified therein, we have characterized in detail three distinct α/ß-hydrolases (LIPESV12_9, LIPESV12_24, LIPESV12_26) and one new α-arabinopyranosidase (GLV12_5). All LIPESV12 enzymes revealed distinct substrate specificities tested against 43 structurally diverse esters and 4 p-nitrophenol carboxyl esters. Of 16 different glycosides tested, the GLV12_5 hydrolysed only p-nitrophenol-α-(L)-arabinopyranose with a high specific activity of about 2.7 kU/mg protein. Most of the α/ß-hydrolases were thermophilic and revealed a high tolerance to, and high activities in the presence of, numerous heavy metal ions. Among them, the LIPESV12_24 was the best temperature-adapted, retaining its activity after 40 min of incubation at 90 °C. Furthermore, enzymes were active in organic solvents (e.g., >30% methanol). Both LIPESV12_24 and LIPESV12_26 had the GXSXG pentapeptides and the catalytic triads Ser-Asp-His typical to the representatives of carboxylesterases of EC 3.1.1.1.

Expression and characterization of a new isoform of the 9 kDa allergenic lipid transfer protein from tomato (variety San Marzano).

Lipid transfer proteins (LTPs) are food allergens found first in fruits of the Rosaceae family and later identified in other food plants. Their high structural stability causes them to behave as allergens in cooked and processed foods. Allergenic LTPs have been identified in tomato fruits as well, but studies of their thermal stability and structural characteristics are limited. In this article we report the identification of the coding region for a novel 9 kDa LTP isoform in the tomato variety San Marzano, together with the expression of the recombinant mature protein. The purified recombinant protein was further characterized for its thermal stability and was found to bind 1-palmitoil-2-lysophosphatidylcholine (Lyso-C16) after thermal treatments up to 105 °C. Analysis of a modeling derived structure of the protein allowed the identification of possible epitope regions on the molecular surface.

Draft genome sequence of Sphingobium sp. strain ba1, resistant to kanamycin and nickel ions.

The genome sequence of a Sphingobium strain capable of tolerating high concentrations of Ni ions, and exhibiting natural kanamycin resistance, is presented. The presence of a transposon derived kanamycin resistance gene and several genes for efflux-mediated metal resistance may explain the observed characteristics of the new Sphingobium isolate.

Overview of plant chitinases identified as food allergens.

Food allergies are induced by proteins belonging to a limited number of families. Unfortunately, relationships between protein structure and capacity to induce the immune response have not been completely clarified yet, which precludes possible improvements in the diagnosis, prevention, and therapy of allergies. Plant chitinases constitute a good example of food allergenic proteins for which structural analysis of allergenicity has only been carried out partially. In plants, there are at least five structural classes of chitinases plus a number of chitinase-related polypeptides. Their allergenicity has been mostly investigated for chitinases of class I, due to both their higher prevalence among plant chitinases and by the high structural similarity between their substrate-binding domain and hevein, a well-known allergen present in the latex of rubber trees. Even if allergenic molecules have been identified for at least three other classes of plant chitinases, the involvement of the different structural motifs in the allergenicity of molecules has been disregarded so far. In this review, we provide a structurally based catalog of plant chitinases investigated for allergenicity, which could be a useful base for further studies aimed at better clarifying the structure-allergenicity relationships for this protein family.

Rhodobacter sphaeroides adaptation to high concentrations of cobalt ions requires energetic metabolism changes.

Rhodobacter sphaeroides has for a long time been investigated for its adaptive capacities to different environmental and nutritional conditions, including presence of heavy metals, which make it a valuable model organism for understanding bacterial adaptation to metal stress conditions and future environmental applications, such as bioremediation of polluted sites. To further characterize the capability of R. sphaeroides to cope with high cobalt ion concentrations, we combined the selection of adaptive defective mutants, carried out by negative selection of transposon insertional libraries on 5 mM Co(2+) -enriched solid medium, with the analysis of growing capacities and transcriptome profiling of a selected mutant (R95). A comparative analysis of results from the mutant and wild-type strains clearly indicated that the adaptive ability of R. sphaeroides strongly relies on its ability to exploit any available energy-supplying metabolisms, being able to behave as photo- or chemotrophic microorganism. The selected R95 mutant, indeed, exhibits a severe down-expression of an ABC sugar transporter, which results nonpermissive for its growth in cobalt-enriched media under aerobic conditions. Interestingly, the defective expression of the transporter does not have dramatic effects on the growth ability of the mutant when cultivated under photosynthetic conditions.

Genome walking by next generation sequencing approaches.

Genome Walking (GW) comprises a number of PCR-based methods for the identification of nucleotide sequences flanking known regions. The different methods have been used for several purposes: from de novo sequencing, useful for the identification of unknown regions, to the characterization of insertion sites for viruses and transposons. In the latter cases Genome Walking methods have been recently boosted by coupling to Next Generation Sequencing technologies. This review will focus on the development of several protocols for the application of Next Generation Sequencing (NGS) technologies to GW, which have been developed in the course of analysis of insertional libraries. These analyses find broad application in protocols for functional genomics and gene therapy. Thanks to the application of NGS technologies, the original vision of GW as a procedure for walking along an unknown genome is now changing into the possibility of observing the parallel marching of hundreds of thousands of primers across the borders of inserted DNA molecules in host genomes.

A protein shuttle system to target RNA into mitochondria.

Mitochondria play a key role in essential cellular functions. A deeper understanding of mitochondrial molecular processes is hampered by the difficulty of incorporating foreign nucleic acids into organelles. Mitochondria of most eukaryotic species import cytosolic tRNAs. Based on this natural process, we describe here a powerful shuttle system to internalize several types of RNAs into isolated mitochondria. We demonstrate that this tool is useful to investigate tRNA processing or mRNA editing in plant mitochondria. Furthermore, we show that the same strategy can be used to address both tRNA and mRNA to isolated mammalian mitochondria. We anticipate our novel approach to be the starting point for various studies on mitochondrial processes. Finally, our study provides new insights into the mechanism of RNA import into mitochondria.

Plant mitochondria use two pathways for the biogenesis of tRNAHis.

All tRNA(His) possess an essential extra G(-1) guanosine residue at their 5' end. In eukaryotes after standard processing by RNase P, G(-1) is added by a tRNA(His) guanylyl transferase. In prokaryotes, G(-1) is genome-encoded and retained during maturation. In plant mitochondria, although trnH genes possess a G(-1) we find here that both maturation pathways can be used. Indeed, tRNA(His) with or without a G(-1) are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNA(His) precursors at both positions G(+1) and G(-1). The G(-1) is essential for recognition by plant mitochondrial histidyl-tRNA synthetase. Whether, as shown in prokaryotes and eukaryotes, the presence of uncharged tRNA(His) without G(-1) has a function or not in plant mitochondrial gene regulation is an open question. We find that when a mutated version of a plant mitochondrial trnH gene containing no encoded extra G is introduced and expressed into isolated potato mitochondria, mature tRNA(His) with a G(-1) are recovered. This shows that a previously unreported tRNA(His) guanylyltransferase activity is present in plant mitochondria.

Application of a genome walking method for the study of the spinach Lhcb1 multigene family.

We describe the application of a novel genome walking (GW) strategy for the one-shot identification of members of multigene families. The method was used to study the spinach Lhcb1 family (encoding the light harvesting complex protein Lhcb1), for which three cDNAs were known. Two additional genes and regulatory regions of the five members of the family were identified. For one of the newly detected genes, sequencing of full-length cDNA and analysis of regulatory regions by gel-shift are also reported. To our best knowledge, this is the first report on the use of a GW approach for the study of a multigene family.

Mapping of 5' and 3'-ends of sunflower mitochondrial nad6 mRNAs reveals a very complex transcription pattern which includes primary transcripts lacking 5'-UTR.

Nad6 orf, encoded in the sunflower mitochondrial genome in single copy contains a non-conserved 3'-extension. The transcription of the nad6 locus generates a highly complex pattern. Three main transcripts of 1240, 960 and 870 nt have been characterized by different approaches. The two smaller ones are apparently the most abundant of the steady-state RNA population and are generated as primary transcription products as well as by processing of the 1240 nt transcript. Their 5'-UTRs are absent or very short. Whereas the 3'-ends of the 960 nt transcripts contain a TGA codon the shorter one terminates at positions excluding the stop codon. The fate of transcripts to promote the synthesis of NAD6 sunflower protein seems, thus, to rely on the occurrence of mechanisms yet to be identified.

Accumulation and erosion of Mars' south polar layered deposits.

Mars' polar regions are covered with ice-rich layered deposits that potentially contain a record of climate variations. The sounding radar SHARAD on the Mars Reconnaissance Orbiter mapped detailed subsurface stratigraphy in the Promethei Lingula region of the south polar plateau, Planum Australe. Radar reflections interpreted as layers are correlated across adjacent orbits and are continuous for up to 150 kilometers along spacecraft orbital tracks. The reflectors are often separated into discrete reflector sequences, and strong echoes are seen as deep as 1 kilometer. In some cases, the sequences are dipping with respect to each other, suggesting an interdepositional period of erosion. In Australe Sulci, layers are exhumed, indicating recent erosion.

Comparison of promoters controlling on the sunflower mitochondrial genome the transcription of two copies of the same native trnK gene reveals some differences in their structure.

Two copies of native trnK (UUU) gene are encoded on the sunflower mitochondrial DNA. They lie within two 12-kb direct repeats, presumably generated by a duplication event. During an investigation aimed at detecting DNA regions activating the trnK1 and trnK2 genes, three distinct promoters have been identified. Their locations were deduced using standard procedures (RT-PCR, RNA capping and 5'RACE) usually employed for the detection of transcription initiation sites (TISs). Promoters P3 and P2 control two independent partially overlapping transcription units containing the trnK2 and ccb206 genes, respectively. Promoter P1 has been mapped about 5200 bp upstream of the trnK1 gene which is part of a transcription unit also containing exons c, d and e of the nad2 gene, 5' to the tRNA gene. Most probably this promoter is not alone in controlling this transcription unit because this DNA region could be cotranscribed, at least partially, starting from other two promoters located upstream of the trnC and trnN genes, respectively. These genes have been previously mapped in a 5' region adjacent to the cluster containing nad2 exons c, d and e and the trnK1 gene. The comparative analysis of promoters P3 and P1 suggests that the difference between them could be related to the duplication event generating the second copy of trnK gene. The availability of a high number of new promoters belonging to dicot mitochondrial genomes makes possible to note some of their specific features.