High-resolution structural insights into the heliorhodopsin family.

Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), has recently been discovered. Unlike in the known rhodopsins, in HeRs the N termini face the cytoplasm. The function of HeRs remains unknown. We present the structures of the bacterial HeR-48C12 in two states at the resolution of 1.5 Å, which highlight its remarkable difference from all known rhodopsins. The interior of HeR's extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (Schiff base cavity [SBC]) surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH, a planar triangular molecule (acetate) is present in the SBC. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs, suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement in fundamental redox biological processes.

Charting the diversity of uncultured viruses of Archaea and Bacteria.

BACKGROUND: Viruses of Archaea and Bacteria are among the most abundant and diverse biological entities on Earth. Unraveling their biodiversity has been challenging due to methodological limitations. Recent advances in culture-independent techniques, such as metagenomics, shed light on the unknown viral diversity, revealing thousands of new viral nucleotide sequences at an unprecedented scale. However, these novel sequences have not been properly classified and the evolutionary associations between them were not resolved. RESULTS: Here, we performed phylogenomic analysis of nearly 200,000 viral nucleotide sequences to establish GL-UVAB: Genomic Lineages of Uncultured Viruses of Archaea and Bacteria. The pan-genome content of the identified lineages shed light on some of their infection strategies, potential to modulate host physiology, and mechanisms to escape host resistance systems. Furthermore, using GL-UVAB as a reference database for annotating metagenomes revealed elusive habitat distribution patterns of viral lineages and environmental drivers of community composition. CONCLUSIONS: These findings provide insights about the genomic diversity and ecology of viruses of prokaryotes. The source code used in these analyses is freely available at https://sourceforge.net/projects/gluvab/.

New viral biogeochemical roles revealed through metagenomic analysis of Lake Baikal.

BACKGROUND: Lake Baikal is the largest body of liquid freshwater on Earth. Previous studies have described the microbial composition of this habitat, but the viral communities from this ecosystem have not been characterized in detail. RESULTS: Here, we describe the viral diversity of this habitat across depth and seasonal gradients. We discovered 19,475 bona fide viral sequences, which are derived from viruses predicted to infect abundant and ecologically important taxa that reside in Lake Baikal, such as Nitrospirota, Methylophilaceae, and Crenarchaeota. Diversity analysis revealed significant changes in viral community composition between epipelagic and bathypelagic zones. Analysis of the gene content of individual viral populations allowed us to describe one of the first bacteriophages that infect Nitrospirota, and their extensive repertoire of auxiliary metabolic genes that might enhance carbon fixation through the reductive TCA cycle. We also described bacteriophages of methylotrophic bacteria with the potential to enhance methanol oxidation and the S-adenosyl-L-methionine cycle. CONCLUSIONS: These findings unraveled new ways by which viruses influence the carbon cycle in freshwater ecosystems, namely, by using auxiliary metabolic genes that act upon metabolisms of dark carbon fixation and methylotrophy. Therefore, our results shed light on the processes through which viruses can impact biogeochemical cycles of major ecological relevance. Video Abstract.

RISSC: a novel database for ribosomal 16S-23S RNA genes spacer regions.

A novel database, under the acronym RISSC (Ribosomal Intergenic Spacer Sequence Collection), has been created. It compiles more than 1600 entries of edited DNA sequence data from the 16S-23S ribosomal spacers present in most prokaryotes and organelles (e.g. mitochondria and chloroplasts) and is accessible through the Internet (http://ulises.umh.es/RISSC), where systematic searches for specific words can be conducted, as well as BLAST-type sequence searches. Additionally, a characteristic feature of this region, the presence/absence and nature of tRNA genes within the spacer, is included in all the entries, even when not previously indicated in the original database. All these combined features could provide a useful documentation tool for studies on evolution, identification, typing and strain characterization, among others.

[Taxonomical study of bacteria from genera Alteromonas and Pseudoalteromonas isolated from Black Sea water and invertebrates].

Nineteen (19) strains of bacteria have been isolated from the Black Sea water and invertebrates (mollusks and actinia). Most of them have been identified as Alteromonas macleodii, Pseudoalteromonas citrea and P. haloplanktis on the basis of polyphasic taxonomical analysis. Six strains showed 96-97 % similarity to 16S rRNA sequence of the known species of Pseudoalteromonas and obviously belonged to new species. The studied strains have been characterized by a wide spectrum of phenotypic features (morphology, enzyme activity, spectra of carbon nutrition, antibiotic sensitivity); high sensitivity of P. haloplanktis strains to cephalotin and resistance of A. macleodii strains to furadonin made them different than other studied strains of Alteromonas and Pseudoalteromonas.

Biotechnological potential of halobacteria.

The extremely halophilic archaebacteria (halobacteria) became an early focus of scientific interest owing to their role in salted food deterioration. In more recent times their peculiar physiology involving extreme adaptation to the salt environment and other unique features have allowed the development of other applied interests. Their similarities to eukaryotic cells at the level of cell division justifies their use in the prescreening for anti-cancer drugs, and some of their antigens could be used for cancer diagnosis. Their unique retinal proteins can be used as light-biosensors and the use of the purple membrane (pm) as reversible holographic medium has already been developed. Halobacterial enzymes are an extremely tough raw material for enzyme technology, particularly for applications in which the reaction mixture has very low water activity. Thanks to their peculiar lipids and to the production of polysaccharides by some halobacteria, their cultures could be used for enhanced oil recovery. Some halobacteria are excellent producers of industrially interesting biopolymers. The use of halobacteria as producers of polyhydroxyalkanoates, biological polyesters such as poly-3-hydroxybutyrate, with the properties of biodegradable thermoplastics, is being considered.

Application of four molecular techniques for typing outbreak-associated Mycobacterium tuberculosis strains.

We applied four molecular techniques for the typing of strains of Mycobacterium tuberculosis associated with outbreaks: RFLP of the IS6110 insertion sequence, spoligotyping, RAPD, and PCR-IS6110. All 4 techniques were applied to 18 strains which were shown by epidemiological data to be involved in 6 outbreaks. All the methods classified the strains into the same groups as the classical epidemiological data did, but RFLP of the IS6110 insertion sequence and spoligotyping are laborious techniques requiring more than a full day's work, whilst RAPD and PCR IS6110 are simple methods easily incorporated into the daily routine. Nevertheless, a large-scale process of standardization and evaluation is necessary in order to be able to establish the true value of the latter two methods as intraspecific characterization markers for M. tuberculosis isolates.

Application of molecular biology techniques to the diagnosis of nontuberculous mycobacterial infections.

A total of 19,723 clinical samples were cultivated for the detection of mycobacteria from January 1995 to March 2001. The 203 strains of nontuberculous mycobacteria isolated were identified with the use of molecular techniques in combination with traditional biochemical tests. The molecular methods applied were PCR-restriction fragment length polymorphism analysis (PRA) alone or in combination with 16S rRNA and 16S-23S spacer sequencing. The patient records of those with specimens positive for mycobacteria were analysed to evaluate the clinical significance of the culture results. Twenty-five of the 124 patients analysed (20%) were regarded as having clinical mycobacteriosis. The main species associated with mycobacteriosis were: Mycobacterium avium (13 cases), M. intracellulare (2 cases), M. kansasii (5 cases), M. chelonae (2 cases), M. malmoense (1 case), M. scrofulaceum (1 case) and M. marinum (1 case). The use of PRA alone or in combination with gene sequencing provided valuable help in discerning mycobacteria at both the intra- and interspecies level, thus contributing to a faster and more efficient diagnosis and epidemiological follow-up.

The use of arbitrarily primed PCR (AP-PCR) to develop taxa specific DNA probes of known sequence.

A rapid polymerase chain reaction (PCR) method to obtain taxa-specific DNA probes has been developed. The oligonucleotide probes derived from the sequences of species-specific (or other taxa) random amplified polymorphic DNA (RAPD) fragments. The methodology was applied to design probes for the halophilic archaeal species Haloferax mediterranei. With this technique, DNA probes of known sequence can be generated easily and without any previous knowledge about the properties of the microorganisms.

Use of thiobacillus ferrooxidans in a coupled microbiological-electrochemical system for wastewater detoxification

We have developed a mixed system, electrochemical-microbiological, that can be used for detoxifying organic compounds present in wastewater. In this system, organic matter oxidation takes place at the anode of an electrochemical reactor while ferric iron reduction takes place at the cathode. We have used a growing culture of Thiobacillus ferrooxidans to regenerate the ferric ions consumed. The culture is used as the catholyte (solution in the cathode compartment) of the system and is therefore permanently subjected to an electric field. We have verified that, under our working conditions, the culture is able to oxidize ferrous ions for long periods of time (up to 15 days) depending on the intensity of the applied current. We have checked the performance of this system in methanol oxidation. Our results show that it decreases the energy cost by 35% when com- pared with the pure electrochemical system traditionally used. Copyright 1999 John Wiley & Sons, Inc.

Sequence microdiversity at the ribosomal RNA operons of Escherichia coli pyelonephritogenic strains.

OBJECTIVE: To determine whether Escherichia coli strains isolated from patients with uncomplicated acute pyelonephritis can be distinguished from those isolated from patients with complicated acute pyelonephritis on the basis of the genetic background. METHODS: In total, 103 E. coli strains isolated from patients with acute pyelonephritis (59 uncomplicated pyelonephritis (UAP) and 44 complicated pyelonephritis (CAP)) were characterized by RFLP of the intergenic spacer region 16S-23S rRNA, the presence of three alternative sequences found in the polymorphic V6 loop of the 16S rRNA gene, the presence of the pap gene, and antibiotic susceptibility. RESULTS: At similarity levels of 70%, four RFLP groups (alpha1, alpha2, beta1 and beta2) were discerned. Strains from UAP were statistically significant for alpha RFLP, with a strong association with the presence of the pap gene, V6-I sequence and antibiotic multisensitivity. Strains from CAP randomly belonged to the alpha or beta RFLP groups, with a very low presence of the pap gene, and random presence of V6 sequences, and were multiresistant to antibiotics. When the CAP strains were distributed according to underlying pathology, non-obstructive cases had RFLP and V6 polymorphisms similar to those of UAP cases, while obstructive cases were clearly distinct. CONCLUSIONS: UAP and non-obstructive CAP E. coli strains are sensitive to antimicrobials, show a high level of the pap gene and belong to the selective, homogeneous and highly protected molecular alpha2 group, where no recombinations, deletions or insertions are present. On the contrary, obstructive and vesicorenal reflux E. coli strains show significant antimicrobial resistance, high intercistronic heterogenicity (wide presence of block nucleotidic substitutions, deletions or insertions) and significantly lower virulence.

Molecular evidence supporting the existence of two major groups in uropathogenic Escherichia coli.

Molecular methods allow an extremely fine strain typing that can be used to establish the population structure of bacterial species. This methodology has been used to characterize a collection of 74 uropathogenic Escherichia coli obtained from three hospitals located in geographically distant towns in Spain, some representatives of the ECOR collection and other reference strains. Genomic DNA was analyzed by RAPD (Random Amplified Polymorphic DNA) that can characterize a bacterial strain to the level of defining individual clones. The 16S rDNA-23S rDNA spacers were amplified by PCR and submitted to restriction analysis. Finally, the presence or absence of G adhesins in Escherichia coli as well as the type of adhesin (three types are known) have been shown by PCR amplification followed by digestion with restriction enzymes. As expected a wide diversity was shown by RAPD and identical patterns were only found in the case of strains isolated from the same individual, an obvious case of relapse. Analysis of the spacers' restriction patterns showed the presence of two markedly differentiated clusters that we have named alpha and beta. Both RAPD and spacer restriction patterns originated similar clusters of strains showing a consistency in the evolution of the global genome with the sequence variation of the ribosomal spacers. Furthermore, most of the strains having G-adhesin, with only a few exceptions, corresponded to the alpha rRNA spacer group. The two spacer types detected were also consistent with some phenotypic markers such as sucrose and raffinose utilization. The alpha and beta clusters could be intraspecific groups produced by partial sexual isolation or other barriers that are originating a divergent evolution.

Use of the 16S--23S ribosomal genes spacer region in studies of prokaryotic diversity.

The description of microbial diversity by molecular culture-independent techniques most often involves the amplification of the 16S rRNA by PCR gene and either analysis of the diversity of amplified molecules (community fingerprinting) that allows the simultaneous study of many samples or the cloning and sequencing of a significant amount of amplification products. The fact that between the 16S and the 23S genes in the ribosomal operon there is a spacer extremely variable in both sequence and length provides an excellent tool to simplify both approaches. The spacer can be amplified almost as easily as the 16S rDNA taking advantage of conserved nucleotide stretches at the 5' end of the 23S gene and the amplicon can contain different amounts of the 16S rDNA choosing primers at the different conserved areas within this gene. Identified by the acronym RISA (rDNA internal spacer analysis), the spacer addition provides a marker of highly variable size allowing standard separation of the amplification products and the sequence of this hypervariable region is useful in the fine discrimination of operational taxonomic units.

Archaeal Biodiversity in Crystallizer Ponds from a Solar Saltern: Culture versus PCR.

The culturable haloarchaeal diversity in a crystallizer pond from a solar saltern has been analyzed and compared with the biodiversity directly retrieved by analysis of rRNA genes amplified from the environment. Two different sets of culture conditions have been assayed: solid medium with yeast extract as carbon source and liquid media with either yeast extract or a mixture of fishmeal, Spirulina sp., and Artemia salina. Seventeen colonies grown on plates with yeast extract incubated at 30 degrees C were analyzed by 16S rDNA partial sequencing. Sixteen were closely related to haloarchaea of the genus Halorubrum; 13 of them to Halorubrum coriense, a haloarchaeon isolated from a solar saltern pond in Australia, which had not been previously isolated from the pond analyzed in this study; and one to Haloarcula marismortui. Liquid cultures were analyzed by ribosomal internal spacer analysis (RISA) and partial sequencing of the 16SrRNA genes. A total of 18 sequences were analyzed, 15 corresponding to RISA bands obtained from cultures, and 3 from the environmental sample used as inoculum. Thirteen sequences obtained from cultures were related to several Halorubrum species, and 2 to Haloarcula. One of the clones obtained directly from the environmental sample was distantly related to a Natronobacterium, whereas two were related to SPhT, the phylotype most frequently retrieved from this environment by culture independent techniques. Our results show an extremely low diversity for the haloarchaea retrieved by cultivation even when modifications to the standard technique are introduced.

Halobacterium mediterranei spec, nov., a New Carbohydrate-Utilizing Extreme Halophile.

The extremely halophilic rod-shaped bacterium, strain R-4, has features very different from other known Halobacterium species. As do all other extreme halophiles, the organism possesses a high excess of acidic over basic amino acids in its proteins, a cell wall lacking peptidoglycan, and ether-linked diphytanyl lipids. It differs from the "classic" halobacteria in several important ways: it can use many different compounds as sole sources of carbon and energy. It is strongly amylolytic and lipolytic (against different Tweens), and does not produce H(2)S. The cell envelope of R-4 is much thicker than that of other halobacteria. The proportions of polar lipids in the envelope are different, and a new glycolipid sulfate is present in this strain. The envelope has a relatively low protein content, although a glycoprotein similar to the one of Halobacterium salinarium was detected. The G + C content (60%) is lower than that of other halobacteria. It is suggested that R-4 be designated a new species, Halobacterium mediterranei.

Structural studies on the acidic exopolysaccharide from Haloferax denitrificans ATCC 35960.

The structure of a linear, acidic exopolysaccharide isolated from the Archaeon Haloferax denitrificans ATCC 35960 has been determined using NMR spectroscopy. The sugar residues in the repeating unit of the polysaccharide were identified as Gal and GlcA2,3NAc after the assignment of the 1H and 13C resonances using COSY, HOHAHA, HMQC and HMQC-TOCSY experiments. The sequence of the residues in the polysaccharide was established from the inter-residue connectivities observed in the HMQC-NOESY plot. The only sugar released on acid hydrolysis was shown to be D-Gal by GLC analysis, while the absolute configuration of the acidic sugars was shown to be D by comparison of the carbon chemical shifts with those of model compounds. Partial acid hydrolysis yielded a tetrasaccharide, terminated by D-Gal at the reducing end, whose structure confirmed that of the repeating unit of the polysaccharide as-->4)-beta-D-GlcpA2,3NAc-(1-->4)-beta-D-GlcpA2, 3NAc-(1-->4)-alpha-D-GlcpA2,3NAc-(1-->3)-alpha-D-Galp- (1-->, where D-GlcpA2,3NAc is 2,3-diacetamido-2,3-dideoxy-D-glucopyranosiduronic acid.

The structure of the exocellular polysaccharide produced by the Archaeon Haloferax gibbonsii (ATCC 33959).

The structure of the neutral exocellular polysaccharide isolated from the Archaeon Haloferax gibbonsii (ATCC 33959) has been determined using acid hydrolysis, methylation analysis and NMR spectroscopy. The polysaccharide contained D-Man, D-Glc, D-Gal and L-Rha in the ratios 2:1:3:1. The substitution patterns of the sugar residues were deduced from the methylation analysis which indicated the polymer to be composed of a heptasaccharide repeating unit containing two branches. The 1H and 13C NMR resonances of the component sugars were assigned using COSY, HOHAHA, HMQC, and HMQC-TOCSY 2D NMR experiments and the sequence of the sugars in the repeating unit was determined from NOESY and HMBC experiments. The structure can be written as: [formula: see text]

The structure of the exopolysaccharide produced by the halophilic Archaeon Haloferax mediterranei strain R4 (ATCC 33500).

The halophilic Archaeon Haloferax mediterranei exudes into the growth medium a high molecular weight sulfated polysaccharide. The structure of the repeating unit of this polymer was determined by a combination of glycose, methylation, and sulfate analysis, periodate oxidation, and 1D and 2D NMR spectroscopic analysis of the native and periodate-oxidised/reduced polysaccharides. The location of the sulfate group was established from the 1H and 13C NMR data. The structure of the repeating unit of the polysaccharide may be written as [formula: see text]

Intragenomic 16S rDNA divergence in Haloarcula marismortui is an adaptation to different temperatures.

The halophilic archaeon Haloarcula marismortui contains three ribosomal RNA operons, designated rrnA, rrnB, and rrnC. Operons A and C are virtually identical, whereas operon B presents a high divergence in nucleotide sequence, having up to 135 nucleotide polymorphisms among the three 16S, 23S, and 5S ribosomal RNA genes. Quantitative PCR and structural analyses have been performed to elucidate whether the presence of this intragenomic heterogeneity could be an adaptation to the variable environmental conditions in the natural habitat of H. marismortui. Variation in salt concentration did not affect expression but variation in incubation temperature did produce significant changes, with operon B displaying an expression level four times higher than the other two together at 50 degrees C and three times lower at 15 degrees C. We show that the putative promoter region of operon B is also different. In addition, the predicted secondary structure of these genes indicated that they have distinct stabilities at different temperatures and a mutant strain lacking operon B grew slower at high temperatures. This study supports the idea that divergent rRNA genes can be adaptive, with different variants being functional under different environmental conditions (e.g., temperature). The same phenomenon could take place in other halophiles or thermophiles with intragenomic rDNA heterogeneity, where the use of 16S rDNA as a phylogenetic marker and indicator of biodiversity should be used with caution.