Diversity of the cell-wall associated genomic island of the archaeon Haloquadratum walsbyi

Background

Haloquadratum walsbyi represents up to 80 % of cells in NaCl-saturated brines worldwide, but is notoriously difficult to maintain under laboratory conditions. In order to establish the extent of genetic diversity in a natural population of this microbe, we screened a H. walsbyi enriched metagenomic fosmid library and recovered seven novel version of its cell-wall associated genomic island. The fosmid inserts were sequenced and analysed.

Results

The novel cell-wall associated islands delineated two major clades within H. walsbyi. The islands predominantly contained genes putatively involved in biosynthesis of surface layer, genes encoding cell surface glycoproteins and genes involved in envelope formation. We further found that these genes are maintained in the population and that the diversity of this region arises through homologous recombination but also through the action of mobile genetic elements, including viruses.

Conclusions

The population of H. walsbyi in the studied saltern brine is composed of numerous clonal lineages that differ in surface structures including the cell wall. This type of variation probably reflects a number of mechanisms that minimize the infection rate of predating viruses.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1794-8) contains supplementary material, which is available to authorized users.     

Reconstructing viral genomes from the environment using fosmid clones: the case of haloviruses

Background

Metaviriomes, the viral genomes present in an environment, have been studied by direct sequencing of the viral DNA or by cloning in small insert libraries. The short reads generated by both approaches make it very difficult to assemble and annotate such flexible genomic entities. Many environmental viruses belong to unknown groups or prey on uncultured and little known cellular lineages, and hence might not be present in databases.

Methodology and Principal Findings

Here we have used a different approach, the cloning of viral DNA into fosmids before sequencing, to obtain natural contigs that are close to the size of a viral genome. We have studied a relatively low diversity extreme environment: saturated NaCl brines, which simplifies the analysis and interpretation of the data. Forty-two different viral genomes were retrieved, and some of these were almost complete, and could be tentatively identified as head-tail phages (Caudovirales).

Conclusions and Significance

We found a cluster of phage genomes that most likely infect Haloquadratum walsbyi, the square archaeon and major component of the community in these hypersaline habitats. The identity of the prey could be confirmed by the presence of CRISPR spacer sequences shared by the virus and one of the available strain genomes. Other viral clusters detected appeared to prey on the Nanohaloarchaea and on the bacterium Salinibacter ruber, covering most of the diversity of microbes found in this type of environment. This approach appears then as a viable alternative to describe metaviriomes in a much more detailed and reliable way than by the more common approaches based on direct sequencing. An example of transfer of a CRISPR cluster including repeats and spacers was accidentally found supporting the dynamic nature and frequent transfer of this peculiar prokaryotic mechanism of cell protection.     

Rosmarinus officinalis L. essential oils from Spain: composition,antioxidant capacity,lipoxygenase and acetylcholinesterase inhibitory capacities,and antimicrobial activities

Abstract

Six Rosmarinus officinalis L. essential oils (RoEOs) from Murcia (Spain) were studied using gas chromatography. Analysis of their relative and absolute composition showed 1,8-cineole, camphor, α-pinene and camphene to be the main compounds. Moreover, enantioselective gas chromatographies showed different enantiomeric ratios of camphor, limonene and borneol. The antioxidant and chelating capacities of RoEOs and their individual components were measured in an attempt to ascertain the cause of RoEO activity as a whole. All the essential oils were able to inhibit lipoxygenase activity due to bornyl acetate, camphor, terpinen-4-ol and 1,8-cineole and acetylcholinesterase activity due to 3-carene and 1,8-cineole, mainly. When the antimicrobial activities were tested against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans, the least sensitive microorganism was P. aeruginosa and the most sensitive C. albicans. This study furthers our knowledge of six RoEOs from two chemotypes, all of which show potential for industrial applications.     

Determination of whether quorum quenching is a common activity in marine bacteria by analysis of cultivable bacteria and metagenomic sequences

The abundance of quorum quenching (QQ) activity was evaluated in cultivable bacteria obtained from oceanic and estuarine seawater and compared with the frequency of QQ enzyme sequences in the available marine metagenomic collections. The possible role of the high QQ activity found among marine bacteria is discussed.     

Prokaryotic and viral community of the sulfate-rich crust from Peñahueca ephemeral lake,an astrobiology analogue

Peñahueca is an athalassohaline hypersaline inland ephemeral lake originated under semiarid conditions in the central Iberian Peninsula (Spain). Its chemical composition makes it extreme for microbial life as well as a terrestrial analogue of other planetary environments. To investigate the persistence of microbial life associated with sulfate-rich crusts, we applied cultivation-independent methods (optical and electron microscopy, 16S rRNA gene profiling and metagenomics) to describe the prokaryotic community and its associated viruses. The diversity for Bacteria was very low and was vastly dominated by endospore formers related to Pontibacillus marinus of the Firmicutes phylum. The archaeal assemblage was more diverse and included taxa related to those normally found in hypersaline environments. Several ‘metagenome assembled genomes’ were recovered, corresponding to new species of Pontibacillus, several species from the Halobacteria and one new member of the Nanohaloarchaeota. The viral assemblage, although composed of the morphotypes typical of high salt systems, showed little similarity to previously isolated/reconstructed halophages. Several putative prophages of Pontibacillus and haloarchaeal hosts were identified. Remarkably, the Peñahueca sulfate-rich metagenome contained CRISPR-associated proteins and repetitions which were over 10-fold higher than in most hypersaline systems analysed so far.     

The Arabidopsis nitrate transporter NRT2.4 plays a double role in roots and shoots of nitrogen-starved plants

Plants have evolved a variety of mechanisms to adapt to N starvation. NITRATE TRANSPORTER2.4 (NRT2.4) is one of seven NRT2 family genes in Arabidopsis thaliana, and NRT2.4 expression is induced under N starvation. Green fluorescent protein and β-glucuronidase reporter analyses revealed that NRT2.4 is a plasma membrane transporter expressed in the epidermis of lateral roots and in or close to the shoot phloem. The spatiotemporal expression pattern of NRT2.4 in roots is complementary with that of the major high-affinity nitrate transporter NTR2.1. Functional analysis in Xenopus laevis oocytes and in planta showed that NRT2.4 is a nitrate transporter functioning in the high-affinity range. In N-starved nrt2.4 mutants, nitrate uptake under low external supply and nitrate content in shoot phloem exudates was decreased. In the absence of NRT2.1 and NRT2.2, loss of function of NRT2.4 (triple mutants) has an impact on biomass production under low nitrate supply. Together, our results demonstrate that NRT2.4 is a nitrate transporter that has a role in both roots and shoots under N starvation.     

CO Dehydrogenase Genes Found in Metagenomic Fosmid Clones from the Deep Mediterranean Sea

The use of carbon monoxide (CO) as a biological energy source is widespread in microbes. In recent years, the role of CO oxidation in superficial ocean waters has been shown to be an important energy supplement for heterotrophs (carboxydovores). The key enzyme CO dehydrogenase was found in both isolates and metagenomes from the ocean''s photic zone, where CO is continuously generated by organic matter photolysis. We have also found genes that code for both forms I (low affinity) and II (high affinity) in fosmids from a metagenomic library generated from a 3,000-m depth in the Mediterranean Sea. Analysis of other metagenomic databases indicates that similar genes are also found in the mesopelagic and bathypelagic North Pacific and on the surfaces of this and other oceanic locations (in lower proportions and similarities). The frequency with which this gene was found indicates that this energy-generating metabolism would be at least as important in the bathypelagic habitat as it is in the photic zone. Although there are no data about CO concentrations or origins deep in the ocean, it could have a geothermal origin or be associated with anaerobic metabolism of organic matter. The identities of the microbes that carry out these processes were not established, but they seem to be representatives of either Bacteroidetes or Chloroflexi.Carbon monoxide (CO) oxidation is a source of energy for a wide diversity of prokaryotes and is an important process within the global carbon cycle. There is a wide diversity of CO oxidation pathways among both archaea and bacteria (27, 28), and their wide distribution attests to both the ecological importance and ancient origin of CO oxidation. Most of these pathways are anaerobic (31, 40) and have been reported in both archaea and bacteria. However, aerobic CO oxidation is found only in a few groups of bacteria, specifically in many Actinobacteria and Proteobacteria spp. and in at least one Firmicutes sp. (for examples, see references 16, 17, 26, 35, 46, and 47). Classically, aerobic oxidation of CO has been known to be carried out in soils where, in addition to geological or anthropogenic emissions, there are local biological sources connected to plant roots and animals (15, 18, 19). However, more recently, the relevance of CO oxidation processes in the marine environment has also become clear, mostly from evidence from the fields of genomics and metagenomics (26, 42, 43).The aerobic oxidation of CO is very amenable to genomic analysis, since the genes involved are very characteristic, and their presence in marine bacterial genomes and in metagenomic databases can be considered diagnostic. The genes required for aerobic CO oxidation were first described in detail in chemolithoautotrophic Oligotropha carboxidovorans OM5 (10, 35, 36). The enzyme CO dehydrogenase (CODH) catalyzes the oxidation of CO and water to produce carbon dioxide, two electrons, and two protons (8, 11). The electrons are transferred to an electron transfer chain and used to generate a proton gradient across the membrane. Three genes, coxL, coxM, and coxS (for large, medium, and small subunits, respectively), encode the polypeptides for the CODH enzyme. Two heterotrimers, each composed of one CoxL, CoxM, and CoxS subunit, combine to form a functional aerobic CODH enzyme. The large subunit contains the molybdenum cofactor, the medium subunit binds flavin adenine dinucleotide, and the small subunit has two iron-sulfur clusters (13). In addition to these three genes, a number of other accessory genes have also been identified (CoxB, CoxC, CoxH, CoxD, CoxE, CoxF, CoxG, CoxI, and CoxK) that are believed to be required in the processes of regulation, posttranslational modification, and anchorage of the CODH complex to the cytoplasmic membrane. A number of these accessory genes are membrane-bound proteins themselves (CoxB, CoxC, CoxH, and CoxK), containing several transmembrane helices, and indeed, in O. carboxidovorans OM5, the CODH enzyme itself has been observed to associate with the inner cytoplasmic membrane.Based on sequence differences, genome organization, and catalytic properties, there are two types of aerobic molybdenum-based CODH (the anaerobic enzymes are a different class of genes) (20). Both forms can be readily differentiated from other molybdenum hydroxylases by phylogenetic analysis. Form I CODH (also called OMS, named after Oligotropha, Mycobacterium, and Pseudomonas) has been conclusively proven by mutagenesis experiments and X-ray crystallography (8, 32, 35) to be the key enzyme in aerobic CO oxidation by carboxydotrophic bacteria, i.e., those that can grow on CO as the sole carbon and energy source (at >10% CO concentration). The reaction mechanism has also been clearly defined. Form I CODH large-subunit CoxL can be readily diagnosed by its characteristic catalytic site motif AYXCSFR. Moreover, in all the organisms in which form I CODH genes have been identified so far, the genomic organization of the three subunits is always M→S→L. The organization of the accessory genes, however, may vary from organism to organism.There is much less known about the other form, form II CODH (or BMS, after Bradyrhizobium, Mesorhizobium, and Sinorhizobium), which was first described in Bradyrhizobium japonicum USDA 110 (23), a gram-negative bacterial strain and a nitrogen-fixing symbiont of soybeans. Form II CODH enables these bacteria to grow, albeit slowly, in the presence of CO as the sole carbon and energy source, but the rate of CO oxidation by form II CODH of B. japonicum USDA 110 is 10 to 1,000 times lower than that for form I CODH in O. carboxidovorans OM5 and Pseudomonas carboxydohydrogena OM5. The catalytic site of the form II CoxL large subunit is AYRGAGR. The genome organization of the form II subunits is S→L→M, different from that of form I. The number of accessory genes present along with these genes is also variable (20). Form II is often found as a paralogous copy of three subunits of form I, but without the accompanying set of CODH-related genes. This is not surprising, since in most cases, the genes appear to be already associated with the form I cluster elsewhere in the genome, like in Rhodothermus marinus DSM 4252, Dinoroseobacter shibae DFL 12, and Bradyrhizobium sp. strain BTAi1.The discovery of the role played by CODH in marine waters is relatively recent. First, it was found in the genome of Silicibacter pomeroyi DSS-3, a marine alphaproteobacterium of the Roseobacter cluster (26), which was concomitantly proven to be able to oxidize CO at low concentrations (as should be expected in marine waters). Later on the process, it was also found in metagenomic studies of surface waters for the Sargasso Sea metagenome project (26, 45). It has been proposed that many heterotrophic bacteria in surface waters are lithoheterotrophs and take advantage of the CO released by organic matter photolysis as an alternative energy source to supplement the scarce dissolved organic matter in a way akin to the photoheterotrophy mediated by proteorhodopsin or anoxygenic photosynthesis.We recently found evidence of a CODH presence deep in the Mediterranean Sea by the end sequencing of fosmids from a metagenomic library from a 3,000-m depth in the Ionian Sea (southeast of Sicily, Italy) (24). Here we present the analysis of nine fully sequenced fosmids that were chosen on the basis of the presence of CODH cluster genes at their ends. The results confirm the presence of complete CODH clusters, including one that has the gene sequence and cluster structure of a form I CODH. Although the source of CO deep in the ocean is unclear, the frequency in which these genes were found and the retrieval of similar sequences from the deep-ocean metagenomic database of the Hawaii Ocean Time-Series (HOT) station (7, 21) point toward an important contribution of this lithotrophic metabolism deep in the ocean, similarly relevant to that found in the surface waters.     

Complete Genome Sequence of the Copiotrophic Marine Bacterium Alteromonas macleodii Strain ATCC 27126T

The genome of Alteromonas macleodii strain ATCC 27126^T has been resequenced and closed into a single contig. We describe here the genome of this important and globally distributed marine bacterium.     

Quorum quenching in cultivable bacteria from dense marine coastal microbial communities

Acylhomoserine lactone (AHLs)-mediated quorum-sensing (QS) processes seem to be common in the marine environment and among marine pathogenic bacteria, but no data are available on the prevalence of bacteria capable of interfering with QS in the sea, a process that has been generally termed 'quorum quenching' (QQ). One hundred and sixty-six strains isolated from different marine dense microbial communities were screened for their ability to interfere with AHL activity. Twenty-four strains (14.4%) were able to eliminate or significantly reduce N-hexanoyl-l-homoserine lactone activity as detected by the biosensor strain Chromobacterium violaceum CV026, a much higher percentage than that reported for soil isolates, which reinforces the ecological role of QS and QQ in the marine environment. Among these, 15 strains were also able to inhibit N-decanoyl-l-homoserine lactone activity and all of them were confirmed to enzymatically inactivate the AHL signals by HPLC-MS. Active isolates belonged to nine different genera of prevalently or exclusively marine origin, including members of the Alpha- and Gammaproteobacteria (8), Actinobacteria (2), Firmicutes (4) and Bacteroidetes (1). Whether the high frequency and diversity of cultivable bacteria with QQ activity found in near-shore marine isolates reflects their prevalence among pelagic marine bacterial communities deserves further investigation in order to understand the ecological importance of AHL-mediated QS and QQ processes in the marine environment.