The Halobacteria are a well-studied archaeal class and numerous investigations are showing how their diversity is distributed amongst genomes and geographic locations. Evidence indicates that recombination between species continuously facilitates the arrival of new genes, and within species, it is frequent enough to spread acquired genes amongst all individuals in the population. To create permanent independent diversity and generate new species, barriers to recombination are probably required. The data support an interpretation that rates of evolution (e.g., horizontal gene transfer and mutation) are faster at creating geographically localized variation than dispersal and invasion are at homogenizing genetic differences between locations. Therefore, we suggest that recurrent episodes of dispersal followed by variable periods of endemism break the homogenizing forces of intrapopulation recombination and that this process might be the principal stimulus leading to divergence and speciation in Halobacteria.
Haloarchaea; speciation; biogeography; homologous recombination; horizontal gene transfer; population genetics
The aim of this study has been to investigate the potential of serum biomarkers used in clinical practice (CEA, CYFRA 21–1, SCC) together with the serum epidermal growth factor receptor (EGFR) and its associated ligands (EGF, TGF-α, HB-EGF) as outcome predictors of non-small cell lung cancer (NSCLC) patients treated with the TKI erlotinib. The pretreatment levels of these markers were evaluated through immunoassays carried out in 58 patients. The progression-free survival (PFS) and overall survival (OS) were assessed by the Kaplan-Meier method and differences between groups were compared by means of the Log-Rank test. Association of risk factors with survival was evaluated using the univariate and multivariate Cox modelling procedures. Higher CEA (>5 ng/mL) and sEGFR (>56.87 ng/mL) concentrations associated significantly with a higher overall survival. The pre-treatment sEGFR serum levels constituted an independent prognostic factor. The EGFR gene mutational status and the sEGFR level combination was the single to associate significantly with longer progression-free survival periods, in circumstances in which the EGFR gene was mutated and increased protein serum levels were detected. The overall survival as assessed through a Cox analysis revealed similar death hazards with respect to low sEGFR levels combined both with non-mutated EGFR genotypes and low CEA serum levels. Our results suggest that the pre-treatment CEA and sEGFR serum levels may provide a comparable source of information to that supplied by the EGFR gene mutational status with respect to the prognosis of erlotinib treated NSCLC patients. A combined sEGFR and CEA level appraisal could be of considerable value to select patients to undergo EGFR-TKI treatments.
Electronic supplementary material
The online version of this article (doi:10.1186/s40064-015-0891-0) contains supplementary material, which is available to authorized users.
Erlotinib; EGFR; CEA; Non-small cell lung cancer; Survival prediction; Prognosis
The outer membrane proteins (OMPs) of Gram-negative bacteria play a crucial role in virulence and pathogenesis. Identification of these proteins represents an important goal for bacterial proteomics, because it aids in vaccine development. Here, we have developed such an approach for Ehrlichia ruminantium, the obligate intracellular bacterium that causes heartwater. A preliminary whole proteome analysis of elementary bodies, the extracellular infectious form of the bacterium, had been performed previously, but information is limited about OMPs in this organism and about their role in the protective immune response. Identification of OMPs is also essential for understanding Ehrlichia’s OM architecture, and how the bacterium interacts with the host cell environment. First, we developed an OMP extraction method using the ionic detergent sarkosyl, which enriched the OM fraction. Second, proteins were separated via one-dimensional electrophoresis, and digested peptides were analyzed via nano-liquid chromatographic separation coupled with mass spectrometry (LC-MALDI-TOF/TOF). Of 46 unique proteins identified in the OM fraction, 18 (39%) were OMPs, including 8 proteins involved in cell structure and biogenesis, 4 in transport/virulence, 1 porin, and 5 proteins of unknown function. These experimental data were compared to the predicted subcellular localization of the entire E. ruminantium proteome, using three different algorithms. This work represents the most complete proteome characterization of the OM fraction in Ehrlichia spp. The study indicates that suitable subcellular fractionation experiments combined with straightforward computational analysis approaches are powerful for determining the predominant subcellular localization of the experimentally observed proteins. We identified proteins potentially involved in E. ruminantium pathogenesis, which are good novel targets for candidate vaccines. Thus, combining bioinformatics and proteomics, we discovered new OMPs for E. ruminantium that are valuable data for those investigating new vaccines against this organism. In summary, we provide both pioneering data and novel insights into the pathogenesis of this obligate intracellular bacterium.
Recent metagenomic studies on saltern ponds with intermediate salinities have determined that their microbial communities are dominated by both Euryarchaeota and halophilic bacteria, with a gammaproteobacterium closely related to the genera Alkalilimnicola and Arhodomonas being one of the most predominant microorganisms, making up to 15% of the total prokaryotic population. Here we used several strategies and culture media in order to isolate this organism in pure culture. We report the isolation and taxonomic characterization of this new, never before cultured microorganism, designated M19-40T, isolated from a saltern located in Isla Cristina, Spain, using a medium with a mixture of 15% salts, yeast extract, and pyruvic acid as the carbon source. Morphologically small curved cells (young cultures) with a tendency to form long spiral cells in older cultures were observed in pure cultures. The organism is a Gram-negative, nonmotile bacterium that is strictly aerobic, non-endospore forming, heterotrophic, and moderately halophilic, and it is able to grow at 10 to 25% (wt/vol) NaCl, with optimal growth occurring at 15% (wt/vol) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that strain M19-40T has a low similarity with other previously described bacteria and shows the closest phylogenetic similarity with species of the genera Alkalilimnicola (94.9 to 94.5%), Alkalispirillum (94.3%), and Arhodomonas (93.9%) within the family Ectothiorhodospiraceae. The phenotypic, genotypic, and chemotaxonomic features of this new bacterium showed that it constitutes a new genus and species, for which the name Spiribacter salinus gen. nov., sp. nov., is proposed, with strain M19-40T (= CECT 8282T = IBRC-M 10768T = LMG 27464T) being the type strain.
Through thermal expansion of oceans and melting of land-based ice, global warming is very likely contributing to the sea level rise observed during the 20th century. The amount by which further increases in global average temperature could affect sea level is only known with large uncertainties due to the limited capacity of physics-based models to predict sea levels from global surface temperatures. Semi-empirical approaches have been implemented to estimate the statistical relationship between these two variables providing an alternative measure on which to base potentially disrupting impacts on coastal communities and ecosystems. However, only a few of these semi-empirical applications had addressed the spurious inference that is likely to be drawn when one nonstationary process is regressed on another. Furthermore, it has been shown that spurious effects are not eliminated by stationary processes when these possess strong long memory. Our results indicate that both global temperature and sea level indeed present the characteristics of long memory processes. Nevertheless, we find that these variables are fractionally cointegrated when sea-ice extent is incorporated as an instrumental variable for temperature which in our estimations has a statistically significant positive impact on global sea level.
We analyzed the prokaryotic community structure of a saltern pond with 21% total salts located in Isla Cristina, Huelva, Southwest Spain, close to the Atlantic ocean coast. For this purpose, we constructed a metagenome (designated as IC21) obtained by pyrosequencing consisting of 486 Mb with an average read length of 397 bp and compared it with other metagenomic datasets obtained from ponds with 19, 33, and 37% total salts acquired from Santa Pola marine saltern, located in Alicante, East Spain, on the Mediterranean coast. Although the salinity in IC21 is closer to the pond with 19% total salts from Santa Pola saltern (designated as SS19), IC21 is more similar at higher taxonomic levels to the pond with 33% total salts from Santa Pola saltern (designated as SS33), since both are predominated by the phylum Euryarchaeota. However, there are significant differences at lower taxonomic levels where most sequences were related to the genus Halorubrum in IC21 and to Haloquadratum in SS33. Within the Bacteroidetes, the genus Psychroflexus is the most abundant in IC21 while Salinibacter dominates in SS33. Sequences related to bacteriorhodopsins and halorhodopsins correlate with the abundance of Haloquadratum in Santa Pola SS19 to SS33 and of Halorubrum in Isla Cristina IC21 dataset, respectively. Differences in composition might be attributed to local ecological conditions since IC21 showed a decrease in the number of sequences related to the synthesis of compatible solutes and in the utilization of phosphonate.
metagenomics; haloarchaea; halophilic bacteria; saltern; prokaryotic diversity
The Halobacteria are known to engage in frequent gene transfer and homologous recombination. For stably diverged lineages to persist some checks on the rate of between lineage recombination must exist. We surveyed a group of isolates from the Aran-Bidgol endorheic lake in Iran and sequenced a selection of them. Multilocus Sequence Analysis (MLSA) and Average Nucleotide Identity (ANI) revealed multiple clusters (phylogroups) of organisms present in the lake. Patterns of intein and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) presence/absence and their sequence similarity, GC usage along with the ANI and the identities of the genes used in the MLSA revealed that two of these clusters share an exchange bias toward others in their phylogroup while showing reduced rates of exchange with other organisms in the environment. However, a third cluster, composed in part of named species from other areas of central Asia, displayed many indications of variability in exchange partners, from within the lake as well as outside the lake. We conclude that barriers to gene exchange exist between the two purely Aran-Bidgol phylogroups, and that the third cluster with members from other regions is not a single population and likely reflects an amalgamation of several populations.
Halobacteria; Multilocus Sequence Analysis (MLSA); Average Nucleotide Identity (ANI); intein; CRISPR
Halobacteria require high NaCl concentrations for growth and are the dominant inhabitants of hypersaline environments above 15% NaCl. They are well-documented to be highly recombinogenic, both in frequency and in the range of exchange partners. In this study, we examine the genetic and genomic variation of cultured, naturally co-occurring environmental populations of Halobacteria. Sequence data from multiple loci (~2500 bp) identified many closely and more distantly related strains belonging to the genera Halorubrum and Haloarcula. Genome fingerprinting using a random priming PCR amplification method to analyze these isolates revealed diverse banding patterns across each of the genera and surprisingly even for isolates that are identical at the nucleotide level for five protein coding sequenced loci. This variance in genome structure even between identical multilocus sequence analysis (MLSA) haplotypes indicates that accumulation of genomic variation is rapid: faster than the rate of third codon substitutions.
Halobacteria; MLSA; genome fingerprinting; Aran-Bidgol lake; environmental population
Colostrum intake by newborn lambs plays a fundamental role in the perinatal period, ensuring lamb survival. In this study, blood plasma samples from two groups of newborn lambs (Colostrum group and Delayed Colostrum group) at 2 and 14 h after birth were treated to reduce the content of high abundance proteins and analyzed using Two-Dimensional Differential in Gel Electrophoresis and MALDI MS/MS for protein identification in order to investigate low abundance proteins with immune function in newborn lambs.
The results showed that four proteins were increased in the blood plasma of lambs due to colostrum intake. These proteins have not been previously described as increased in blood plasma of newborn ruminants by colostrum intake. Moreover, these proteins have been described as having an immune function in other species, some of which were previously identified in colostrum and milk.
In conclusion, colostrum intake modified the low abundance proteome profile of blood plasma from newborn lambs, increasing the concentration of apolipoprotein A-IV, plasminogen, serum amyloid A and fibrinogen, demonstrating that colostrum is essential, not only for the provision of immunoglobulins, but also because of increases in several low abundance proteins with immune function.
Marine salterns are artificial multipond systems designed for the commercial production of salt by evaporation of seawater. We report here the metagenomic sequence of the prokaryotic microbiota of a pond with intermediate salinity (21% total salts) of a saltern located in Isla Cristina, Huelva, southwest Spain.
The clinical management of human brucellosis is still challenging and demands in vitro active antibiotics capable of targeting the pathogen-harboring intracellular compartments. A sustained release of the antibiotic at the site of infection would make it possible to reduce the number of required doses and thus the treatment-associated toxicity. In this study, a hydrophobically modified gentamicin, gentamicin-AOT [AOT is bis(2-ethylhexyl) sulfosuccinate sodium salt], was either microstructured or encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles. The efficacy of the formulations developed was studied both in vitro and in vivo. Gentamicin formulations reduced Brucella infection in experimentally infected THP-1 monocytes (>2-log10 unit reduction) when using clinically relevant concentrations (18 mg/liter). Moreover, in vivo studies demonstrated that gentamicin-AOT-loaded nanoparticles efficiently targeted the drug both to the liver and the spleen and maintained an antibiotic therapeutic concentration for up to 4 days in both organs. This resulted in an improved efficacy of the antibiotic in experimentally infected mice. Thus, while 14 doses of free gentamicin did not alter the course of the infection, only 4 doses of gentamicin-AOT-loaded nanoparticles reduced the splenic infection by 3.23 logs and eliminated it from 50% of the infected mice with no evidence of adverse toxic effects. These results strongly suggest that PLGA nanoparticles containing chemically modified hydrophobic gentamicin may be a promising alternative for the treatment of human brucellosis.
Marine salterns are composed of several shallow ponds with a salinity gradient, from seawater to salt saturation, with gradually changing microbial populations. Here, we report the metagenome sequencing of the prokaryotic microbiota of two ponds with 13% and 33% salinity from a saltern in Santa Pola, Spain.
Thalassosaline waters produced by the concentration of seawater are widespread and common extreme aquatic habitats. Their salinity varies from that of sea water (ca. 3.5%) to saturation for NaCl (ca. 37%). Obviously the microbiota varies dramatically throughout this range. Recent metagenomic analysis of intermediate salinity waters (19%) indicated the presence of an abundant and yet undescribed gamma-proteobacterium. Two strains belonging to this group have been isolated from saltern ponds of intermediate salinity in two Spanish salterns and were named “Spiribacter”.
The genomes of two isolates of “Spiribacter” have been fully sequenced and assembled. The analysis of metagenomic datasets indicates that microbes of this genus are widespread worldwide in medium salinity habitats representing the first ecologically defined moderate halophile. The genomes indicate that the two isolates belong to different species within the same genus. Both genomes are streamlined with high coding densities, have few regulatory mechanisms and no motility or chemotactic behavior. Metabolically they are heterotrophs with a subgroup II xanthorhodopsin as an additional energy source when light is available.
This is the first bacterium that has been proven by culture independent approaches to be prevalent in hypersaline habitats of intermediate salinity (half a way between the sea and NaCl saturation). Predictions from the proteome and analysis of transporter genes, together with a complete ectoine biosynthesis gene cluster are consistent with these microbes having the salt-out-organic-compatible solutes type of osmoregulation. All these features are also consistent with a well-adapted fully planktonic microbe while other halophiles with more complex genomes such as Salinibacter ruber might have particle associated microniches.
Halophilic bacteria; Xanthorhodopsin; Hypersaline; Saltern; Spiribacter; Moderate halophile
Marinobacter lipolyticus strain SM19, isolated from saline soil in Spain, is a moderately halophilic bacterium belonging to the class Gammaproteobacteria. Here, we report the draft genome sequence of this strain, which consists of a 4.0-Mb chromosome and which is able to produce the halophilic enzyme lipase LipBL.
Five strains (1126-1H-08T, 51B-09, 986-08, 1084B-08 and 424-08) were isolated from diseased rainbow trout. Cells were Gram-negative rods, 0.7 µm wide and 3 µm long, non-endospore-forming, catalase and oxidase positive. Colonies were circular, yellow-pigmented, smooth and entire on TGE agar after 72 hours incubation at 25°C. They grew in a temperature range between 15°C to 30°C, but they did not grow at 37°Cor 42°C. Based on 16S rRNA gene sequence analysis, the isolates belonged to the genus Flavobacterium. Strain 1126-1H-08T exhibited the highest levels of similarity with Flavobacterium oncorhynchi CECT 7678T and Flavobacterium pectinovorum DSM 6368T (98.5% and 97.9% sequence similarity, respectively). DNA–DNA hybridization values were 87 to 99% among the five isolates and ranged from 21 to 48% between strain 1126-1H-08T, selected as a representative isolate, and the type strains of Flavobacterium oncorhynchi CECT 7678T and other phylogenetic related Flavobacterium species. The DNA G+C content of strain 1126-1H-08T was 33.2 mol%. The predominant respiratory quinone was MK-6 and the major fatty acids were iso-C15∶0 and C15∶0. These data were similar to those reported for Flavobacterium species. Several physiological and biochemical tests differentiated the novel bacterial strains from related Flavobacterium species. Phylogenetic, genetic and phenotypic data indicate that these strains represent a new species of the genus Flavobacterium, for which the name Flavobacterium plurextorum sp. nov. was proposed. The type strain is 1126-1H-08T ( = CECT 7844T = CCUG 60112T).
Pseudoalteromonas ruthenica strain CP76, isolated from a saltern in Spain, is a moderately halophilic bacterium belonging to the Gammaproteobacteria. Here we report the draft genome sequence, which consists of a 4.0-Mb chromosome, of this strain, which is able to produce the extracellular enzyme haloprotease CPI.
Halomonas titanicae strain BH1 is a heterotrophic, aerobic marine bacterium which was isolated from rusticles of the RMS Titanic wreck. Here we report the draft genome sequence of this halophilic gammaproteobacterium.
We have previously used a de novo metagenomic assembly approach to describe the presence of an abundant gammaproteobacterium comprising nearly 15% of the microbial community in an intermediate salinity solar saltern pond. We have obtained this microbe in pure culture and describe the genome sequencing of the halophilic photoheterotrophic microbe, Spiribacter salinus M19-40.
Prokaryotic diversity in Aran-Bidgol salt lake, a thalasohaline lake in Iran, was studied by fluorescence in situ hybridization (FISH), cultivation techniques, denaturing gradient gel electrophoresis (DGGE) of PCR-amplified fragments of 16S rRNA genes and 16S rRNA gene clone library analysis. Viable counts obtained (2.5–4 × 106 cells mL−1) were similar to total cell abundance in the lake determined by DAPI direct count (3–4×107 cells mL−1). The proportion of Bacteria to Archaea in the community detectable by FISH was unexpectedly high and ranged between 1:3 and 1:2. We analyzed 101 archaeal isolates and found that most belonged to the genera Halorubrum (55%) and Haloarcula (18%). Eleven bacterial isolates obtained in pure culture were affiliated with the genera Salinibacter (18.7%), Salicola (18.7%) and Rhodovibrio (35.3%). Analysis of inserts of 100 clones from the eight 16S rRNA clone libraries constructed revealed 37 OTUs. The majority (63%) of these sequences were not related to any previously identified taxa. Within this sampling effort we most frequently retrieved phylotypes related to Halorhabdus (16% of archaeal sequences obtained) and Salinibacter (36% of bacterial sequences obtained). Other prokaryotic groups that were abundant included representatives of Haloquadratum, the anaerobic genera Halanaerobium and Halocella, purple sulfur bacteria of the genus Halorhodospira and Cyanobacteria.
Archaea; Bacteria; halophilic microorganisms; prokaryotic diversity; hypersaline lake
We describe the microbiota of two hypersaline saltern ponds, one of intermediate salinity (19%) and a NaCl saturated crystallizer pond (37%) using pyrosequencing. The analyses of these metagenomes (nearly 784 Mb) reaffirmed the vast dominance of Haloquadratum walsbyi but also revealed novel, abundant and previously unsuspected microbial groups. We describe for the first time, a group of low GC Actinobacteria, related to freshwater Actinobacteria, abundant in low and intermediate salinities. Metagenomic assembly revealed three new abundant microbes: a low-GC euryarchaeon with the lowest GC content described for any euryarchaeon, a high-GC euryarchaeon and a gammaproteobacterium related to Alkalilimnicola and Nitrococcus. Multiple displacement amplification and sequencing of the genome from a single archaeal cell of the new low GC euryarchaeon suggest a photoheterotrophic and polysaccharide-degrading lifestyle and its relatedness to the recently described lineage of Nanohaloarchaea. These discoveries reveal the combined power of an unbiased metagenomic and single cell genomic approach.
Three halophilic isolates, strains Halo-G*T, AUS-1 and Naxos II, were compared. Halo-G* was isolated from an evaporitic salt crystal from Baja California, Mexico, whereas AUS-1 and Naxos II were isolated from salt pools in Western Australia and the Greek island of Naxos, respectively. Halo-G*T had been exposed previously to conditions of outer space and survived 2 weeks on the Biopan facility. Chemotaxonomic and molecular comparisons suggested high similarity between the three strains. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that the strains clustered with Halorubrum species, showing sequence similarities of 99.2–97.1 %. The DNA–DNA hybridization values of strain Halo-G*T and strains AUS-1 and Naxos II are 73 and 75 %, respectively, indicating that they constitute a single species. The DNA relatedness between strain Halo-G*T and the type strains of 13 closely related species of the genus Halorubrum ranged from 39 to 2 %, suggesting that the three isolates constitute a different genospecies. The G+C content of the DNA of the three strains was 65.5–66.5 mol%. All three strains contained C20C20 derivatives of diethers of phosphatidylglycerol, phosphatidylglyceromethylphosphate and phosphatidylglycerolsulfate, together with a sulfated glycolipid. On the basis of these results, a novel species that includes the three strains is proposed, with the name Halorubrum chaoviator sp. nov. The type strain is strain Halo-G*T (=DSM 19316T =NCIMB 14426T =ATCC BAA-1602T).
Among extremophiles, halophiles are defined as microorganisms adapted to live and thrive in diverse extreme saline environments. These extremophilic microorganisms constitute the source of a number of hydrolases with great biotechnological applications. The interest to use extremozymes from halophiles in industrial applications is their resistance to organic solvents and extreme temperatures. Marinobacter lipolyticus SM19 is a moderately halophilic bacterium, isolated previously from a saline habitat in South Spain, showing lipolytic activity.
Methods and Findings
A lipolytic enzyme from the halophilic bacterium Marinobacter lipolyticus SM19 was isolated. This enzyme, designated LipBL, was expressed in Escherichia coli. LipBL is a protein of 404 amino acids with a molecular mass of 45.3 kDa and high identity to class C β-lactamases. LipBL was purified and biochemically characterized. The temperature for its maximal activity was 80°C and the pH optimum determined at 25°C was 7.0, showing optimal activity without sodium chloride, while maintaining 20% activity in a wide range of NaCl concentrations. This enzyme exhibited high activity against short-medium length acyl chain substrates, although it also hydrolyzes olive oil and fish oil. The fish oil hydrolysis using LipBL results in an enrichment of free eicosapentaenoic acid (EPA), but not docosahexaenoic acid (DHA), relative to its levels present in fish oil. For improving the stability and to be used in industrial processes LipBL was immobilized in different supports. The immobilized derivatives CNBr-activated Sepharose were highly selective towards the release of EPA versus DHA. The enzyme is also active towards different chiral and prochiral esters. Exposure of LipBL to buffer-solvent mixtures showed that the enzyme had remarkable activity and stability in all organic solvents tested.
In this study we isolated, purified, biochemically characterized and immobilized a lipolytic enzyme from a halophilic bacterium M. lipolyticus, which constitutes an enzyme with excellent properties to be used in the food industry, in the enrichment in omega-3 PUFAs.