Inmaculada Meseguer

Production of halocin is a practically universal feature of archaeal halophilic rods

Antagonisms among members of nine phenons of halobacteria were detected by combining two methods based on the double layer technique. Inhibitory activities were not due to phages. The protein nature of the inhibitors indicated that they were halocins. With only one exception, all strains tested exhibited inhibitory activity against other halobacteria due to the production of halocins. A very wide range of activity spectra was detected and the numerical comparative analysis showed little grouping, due to the scarce similarities between them. This indicates that many different halocins are produced by this heterogeneous group of micro‐organisms. Our results show that halocin production should be considered as a practically general feature of halobacteria.

Fine-scale evolution: genomic, phenotypic and ecological differentiation in two coexisting Salinibacter ruber strains

Genomic and metagenomic data indicate a high degree of genomic variation within microbial populations, although the ecological and evolutive meaning of this microdiversity remains unknown. Microevolution analyses, including genomic and experimental approaches, are so far very scarce for non-pathogenic bacteria. In this study, we compare the genomes, metabolomes and selected ecological traits of the strains M8 and M31 of the hyperhalophilic bacterium Salinibacter ruber that contain ribosomal RNA (rRNA) gene and intergenic regions that are identical in sequence and were simultaneously isolated from a Mediterranean solar saltern. Comparative analyses indicate that S. ruber genomes present a mosaic structure with conserved and hypervariable regions (HVRs). The HVRs or genomic islands, are enriched in transposases, genes related to surface properties, strain-specific genes and highly divergent orthologous. However, the many indels outside the HVRs indicate that genome plasticity extends beyond them. Overall, 10% of the genes encoded in the M8 genome are absent from M31 and could stem from recent acquisitions. S. ruber genomes also harbor 34 genes located outside HVRs that are transcribed during standard growth and probably derive from lateral gene transfers with Archaea preceding the M8/M31 divergence. Metabolomic analyses, phage susceptibility and competition experiments indicate that these genomic differences cannot be considered neutral from an ecological perspective. The results point to the avoidance of competition by micro-niche adaptation and response to viral predation as putative major forces that drive microevolution within these Salinibacter strains. In addition, this work highlights the extent of bacterial functional diversity and environmental adaptation, beyond the resolution of the 16S rRNA and internal transcribed spacers regions.

Halocin H6, a Bacteriocin from Haloferax gibbonsii

Summary: Haloferax gibbonsii strain Ma2.39 produces an inhibitor substance designated halocin H6. This halocin was isolated from culture supernatants of the producer strain. It appeared to be a non-inducible bacteriocin with a bactericidal mode of cation and typical ‘single-hit’ kinetics. The protein was purified by a combination of hydroxylapatite affinity chromatography, gel filtration and HPLC. Halocin H6 is a protein of M r 32000; it is heat resistant, non-salt-dependent (it retains its activity after exposure to distilled water) and sensitive to pronase but not to trypsin.

Culture-independent approaches for studying viruses from hypersaline environments

ABSTRACT Hypersaline close-to-saturation environments harbor an extremely high concentration of virus-like particles, but the number of haloviruses isolated so far is still very low. Haloviruses can be directly studied from natural samples by using different culture-independent techniques that include transmission electron microscopy, pulsed-field gel electrophoresis, and different metagenomic approaches. Here, we review the findings of these studies, with a main focus on the metagenomic approaches. The analysis of bulk viral nucleic acids directly retrieved from the environment allows estimations of viral diversity, activity, and dynamics and tentative host assignment. Results point to a diverse and active viral community in constant interplay with its hosts and to a “hypersalineness” quality common to viral assemblages present in hypersaline environments that are thousands of kilometers away from each other.

Effect of halocin H4 on cells of Halobacterium halobium

SUMMARY: The killing of a population of a sensitive strain of Halobacterium halobium by halocin H4 followed exponential kinetics, and the percentage survival of sensitive cells exposed to different concentrations of halocin H4 corresponded to single-hit-type kinetics. Morphological changes were observed in treated cells, which showed swollen, spherical shapes. Halocin H4 affected macromolecule synthesis very little, and only late after the start of the treatment, although the transport of 2-aminoisobutyric acid, a non-metabolizable amino acid, was rapidly stopped. Bacteriorhodopsin-mediated H+ extrusion worked very efficiently in treated cells, and much larger pH decreases were found in treated than in untreated suspensions after illumination, although ATP synthesis was not markedly affected. These findings suggest that the primary target of halocin H4 may be located in the membrane, producing permeability changes and ionic imbalance, which lead to death and cell lysis.

Metatranscriptomic analysis of extremely halophilic viral communities

Hypersaline environments harbour the highest number of viruses reported for aquatic environments. In crystallizer ponds from solar salterns, haloviruses coexist with extremely halophilic Archaea and Bacteria and present a high diversity although little is known about their activity. In this work, we analyzed the viral expression in one crystallizer using a metatranscriptomic approach in which clones from a metaviromic library were immobilized in a microarray and used as probes against total mRNA extracted from the hypersaline community. This approach has two advantages: (i) it overcomes the fact that there is no straightforward, unambiguous way to extract viral mRNA from bulk mRNAs and (ii) it makes the sequencing of all mRNAs unnecessary. Transcriptomic data indicated that the halovirus assemblage was highly active at the time of sampling and the viral groups with the highest expression levels were those related to high GC content haloarchaea and Salinibacter representatives, which are minor components in the environment. Moreover, the changes in the viral expression pattern and in the numbers of free viral particles were analyzed after submitting the samples to two stress conditions: ultraviolet-radiation and dilution. Results showed that Archaea were more sensitive than Bacteria to these stress conditions. The overexpression in the predicted archaeal virus fraction raised and the total numbers of free viruses increased. Furthermore, we identified some very closely related viral clones, displaying single-nucleotide polymorphisms, which were expressed only under certain conditions. These clones could be part of very closely related virus genomes for which we propose the term ‘ecoviriotypes’.

From community approaches to single-cell genomics: the discovery of ubiquitous hyperhalophilic Bacteroidetes generalists

The microbiota of multi-pond solar salterns around the world has been analyzed using a variety of culture-dependent and molecular techniques. However, studies addressing the dynamic nature of these systems are very scarce. Here we have characterized the temporal variation during 1 year of the microbiota of five ponds with increasing salinity (from 18% to >40%), by means of CARD-FISH and DGGE. Microbial community structure was statistically correlated with several environmental parameters, including ionic composition and meteorological factors, indicating that the microbial community was dynamic as specific phylotypes appeared only at certain times of the year. In addition to total salinity, microbial composition was strongly influenced by temperature and specific ionic composition. Remarkably, DGGE analyses unveiled the presence of most phylotypes previously detected in hypersaline systems using metagenomics and other molecular techniques, such as the very abundant Haloquadratum and Salinibacter representatives or the recently described low GC Actinobacteria and Nanohaloarchaeota. In addition, an uncultured group of Bacteroidetes was present along the whole range of salinity. Database searches indicated a previously unrecognized widespread distribution of this phylotype. Single-cell genome analysis of five members of this group suggested a set of metabolic characteristics that could provide competitive advantages in hypersaline environments, such as polymer degradation capabilities, the presence of retinal-binding light-activated proton pumps and arsenate reduction potential. In addition, the fairly high metagenomic fragment recruitment obtained for these single cells in both the intermediate and hypersaline ponds further confirm the DGGE data and point to the generalist lifestyle of this new Bacteroidetes group.

Biopolymer Production by Haloferax Mediterranei

The halobacterium H. mediterranei is an organism of biotechnological potential due to its capacity as polymer producer. This microorganism accumulates poly-s-hydroxybutyrate as intracellular granules in very large amounts. PHB is an interesting biopolymer for its possible use as biodegradable thermoplastic. The conditions of culture for PHB production have been studied and optimized in batch as well as in continuous culture. Phosphate limitation is an essential condition for PHB accumulation in important quantities. Glucose and starch were the carbon sources giving the highest productivities. Under favourable circumstances in batch culture a production of 6.5 g/1 PHB was reached, being 67% of the total biomass dry weight, and with a yield over the carbon source of 0.33 gg-1. There is evidence indicating that the PHB produced by H. mediterranei is a copolymer with monomers of more than four carbon atoms, which is favourable for the manipulation of the physical properties of the polymer. H. mediterranei accumulates another polymer extracellularly. It is a sulfated heteropolysaccharide that has very good rheological properties which are very resistant to environmental stress including, as may be expected, salinity. High productivities of this polysaccharide require sugars as carbon source, a condition that also favours PHB accumulation.

A halocin acting on Na+/H+ exchanger of Haloarchaea as a new type of inhibitor in NHE of mammals

The capability of halocin H6 (a bacteriocin-like protein produced by haloarchaeaHaloferax gibbonsii) to inhibit Na+/H+ exchange (NHE) in mammalian cells and its cardio-protective efficacy on the ischemic and reperfused myocardium were evaluated in the present study. H6 inhibits NHE activity (measured by a flow cytometry method) in a dose-dependent form of cell lines of mammalian origin (HEK293, NIH3T3, Jurkat and HL-1) as well as in primary cell culture from human skeletal muscle (myocytes and fibroblasts).In vivo, an ischemia-reperfusion model in dogs by coronary arterial occlusion was used (two hours of regional ischemia and three hours of reperfusion). In animals treated with halocin H6 there was a significant reduction of premature ventricular ectopic beats and infarct size, whereas blood pressure and heart rate remained unchanged. Up to date, halocin H6 is the only described biological molecule that exerts a, specific inhibitory activity in NHE of eukaryotic cells.ResumenEn el presente trabajo se evalúa la capacidad de la halocina H6 (una proteína tipo bacteriocina producida por la haloarchaeaHaloferax gibbonsii) para inhibir el intercambiador Na+/H+ (NHE) de céludas de mamífero y su posible eficacia cardioprotectora frente a los daños causados por isquemia-reperfusión del miocardio. En experimentosin vitro H6 inhibe la actividad de NHE (determinada por citometría de flujo) de forma dosis-dependiente tanto en líneas celulares de mamíferos (HEK293, NIH3T3, Jurkat y HL-1) como en cultivos primarios de miocitos y fibroblastos aislados de músculo esquelético humano. En experimentosin vivo se utilizó un modelo de isquemia-reperfusión en perros por oclusión de la arteria coronaria (dos horas de isquemia y tres de reperfusión). En animales tratados con halocina H6 se produjo una disminución significativa a nivel estadístico, tanto del número de latidos ectópicos ventriculares como del tamaño del infarto, mientras que no se produjeron cambios tanto en la presión sanguínea como en el ritmo cardíaco. Hasta la fecha la halocina H6 es la única molécula biológica descrita que ejerce una actividad inhibidora específica sobre el NHE de células eucariotas.

Mode of Action of Halocins H4 and H6: Are They Effective Against the Adaptation to High Salt Environments?

Halocins H4 and H6, two bacteriocins produced by halobacterial strains, are proteins able to kill other halobacterial strains than those which produce them. Although their physico-chemical features are quite distinct, their modes of action seem to be similar. Both halocins induce morphological changes and lysis in sensitive cells, affect light-induced pH changes and inhibit α-aminoisobutiric acid transport. All these factors lead us to suppose that the target of both halocins must be located at membrane level, affecting one or more of the mechanisms which take part in the complex machinery of regulation and maintenance of the electrochemical gradients steady state through the membrane. The fact that halobacteria live in extremely aggressive media considerably increases the effectiveness of substances such as halocin H4 and H6 to kill salt-dependent cells.