Extremely halophilic brine community manipulation shows higher robustness of microbiomes inhabiting human-driven solar saltern than naturally driven lake.

Hypersaline ecosystems display taxonomically similar assemblages with low diversities and highly dense accompanying viromes. The ecological implications of viral infection on natural microbial populations remain poorly understood, especially at finer scales of diversity. Here, we sought to investigate the influence of changes in environmental physicochemical conditions and viral predation pressure by autochthonous and allochthonous viruses on host dynamics. For this purpose, we transplanted two microbiomes coming from distant hypersaline systems (solar salterns of Es Trenc in Spain and the thalassohaline lake of Aran-Bidgol lake in Iran), by exchanging the cellular fractions with the sterile-filtered accompanying brines with and without the free extracellular virus fraction. The midterm exposure (1 month) of the microbiomes to the new conditions showed that at the supraspecific taxonomic range, the assemblies from the solar saltern brine more strongly resisted the environmental changes and viral predation than that of the lake. The metagenome-assembled genomes (MAGs) analysis revealed an intraspecific transition at the ecotype level, mainly driven by changes in viral predation pressure, by both autochthonous and allochthonous viruses. IMPORTANCE: Viruses greatly influence succession and diversification of their hosts, yet the effects of viral infection on the ecological dynamics of natural microbial populations remain poorly understood, especially at finer scales of diversity. By manipulating the viral predation pressure by autochthonous and allochthonous viruses, we uncovered potential phage-host interaction, and their important role in structuring the prokaryote community at an ecotype level.

Metagenomics of two aquifers with thermal anomalies in Mallorca Island, and proposal of new uncultivated taxa named following the rules of SeqCode.

Groundwater offers an intriguing blend of distinctive physical and chemical conditions, constituting a challenge for microbial life. In Mallorca, the largest island of Balearic archipelago, harbours a variety of thermal anomalies (i.e., geothermal manifestation where surface aquifers exhibiting temperatures exceeding the regional average). The metagenomes of two aquifers in the centre and southern of the island showed Pseudomonadota to be the most represented phylum when using extracted 16S rRNA gene sequences. However, the microbial structures within and between aquifers were remarkably diverse but similar in their metabolic profiles as revealed by the metagenome-assembled genomes (MAGs) pointing to a prevalence of aerobic chemolithoautotrophic and heterotrophic metabolisms, especially in the Llucmajor aquifer. Also, some evidences of anaerobic lifestyles were detected, which would indicate that these environments either could suffer episodes of oxygen depletion or the anaerobes had been transported from deeper waters. We believe that the local environmental factors (temperature, external inputs or chemistry) seem to be more relevant than the connection and, eventually, transport of microbial cells within the aquifer in determining the highly divergent structures. Notably, most of the reconstructed genomes belonged to undescribed bacterial lineages and from them two high-quality MAGs could be classified as novel taxa named following the rules of the Code for Nomenclature of Prokaryotes Described from Sequence Data (SeqCode). Accordingly, we propose the new species and genus Costitxia debesea gen. nov., sp. nov., affiliated with the novel family Costitxiaceae fam. nov., order Costitxiales ord. nov. and class Costitxiia class. nov.; and the new new species and genus Lloretia debesea gen. nov. sp. nov. affiliated with the novel family Lloretiaceae fam. nov.

Food determines ephemerous and non-stable gut microbiome communities in juvenile wild and farmed Mediterranean fish.

Novel insights were provided by contrasting the composition of wild and farmed fish gut microbiomes because the latter had essentially different environmental conditions from those in the wild. This was reflected in the gut microbiome of the wild Sparus aurata and Xyrichtys novacula studied here, which showed highly diverse microbial community structures, dominated by Proteobacteria, mostly related to an aerobic or microaerophilic metabolism, but with some common shared major species, such as Ralstonia sp. On the other hand, farmed non-fasted S. aurata individuals had a microbial structure that mirrored the microbial composition of their food source, which was most likely anaerobic, since several members of the genus Lactobacillus, probably revived from the feed and enriched in the gut, dominated the communities. The most striking observation was that after a short fasting period (86 h), farmed gilthead seabream almost lost their whole gut microbiome, and the resident community associated with the mucosa had a very much reduced diversity that was highly dominated by a single potentially aerobic species Micrococcus sp., closely related to M. flavus. The results pointed to the fact that, at least for the juvenile S. aurata studied, most of the microbes in the gut were transient and highly dependent on the feed source, and that only after fasting for at least 2 days could the resident microbiome in the intestinal mucosa be determined. Since an important role of this transient microbiome in relation to fish metabolism could not be discarded, the methodological approach needs to be well designed in order not to bias the results. The results have important implications for fish gut studies that could explain the diversity and occasional contradictory results published in relation to the stability of marine fish gut microbiomes, and might provide important information for feed formulation in the aquaculture industry.

Description of two cultivated and two uncultivated new Salinibacter species, one named following the rules of the bacteriological code: Salinibacter grassmerensis sp. nov.; and three named following the rules of the SeqCode: Salinibacter pepae sp. nov., Salinibacter abyssi sp. nov., and Salinibacter pampae sp. nov.

Current -omics methods allow the collection of a large amount of information that helps in describing the microbial diversity in nature. Here, and as a result of a culturomic approach that rendered the collection of thousands of isolates from 5 different hypersaline sites (in Spain, USA and New Zealand), we obtained 21 strains that represent two new Salinibacter species. For these species we propose the names Salinibacter pepae sp. nov. and Salinibacter grassmerensis sp. nov. (showing average nucleotide identity (ANI) values < 95.09% and 87.08% with Sal. ruber M31T, respectively). Metabolomics revealed species-specific discriminative profiles. Sal. ruber strains were distinguished by a higher percentage of polyunsaturated fatty acids and specific N-functionalized fatty acids; and Sal. altiplanensis was distinguished by an increased number of glycosylated molecules. Based on sequence characteristics and inferred phenotype of metagenome-assembled genomes (MAGs), we describe two new members of the genus Salinibacter. These species dominated in different sites and always coexisted with Sal. ruber and Sal. pepae. Based on the MAGs from three Argentinian lakes in the Pampa region of Argentina and the MAG of the Romanian lake Fara Fund, we describe the species Salinibacter pampae sp. nov. and Salinibacter abyssi sp. nov. respectively (showing ANI values 90.94% and 91.48% with Sal. ruber M31T, respectively). Sal. grassmerensis sp. nov. name was formed according to the rules of the International Code for Nomenclature of Prokaryotes (ICNP), and Sal. pepae, Sal. pampae sp. nov. and Sal. abyssi sp. nov. are proposed following the rules of the newly published Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode). This work constitutes an example on how classification under ICNP and SeqCode can coexist, and how the official naming a cultivated organism for which the deposit in public repositories is difficult finds an intermediate solution.

Comparative genome analysis of the genus Hydrotalea and proposal of the novel species Hydrotalea lipotrueae sp. nov., isolated from a groundwater aquifer in the south of Mallorca Island, Spain.

From a collection of > 140 strains isolated from groundwater with thermal anomalies for the purpose of obtaining good candidates with applications in the cosmetic industry, two strains were selected because of their taxonomic novelty. Among the isolates, strains TMF_100T and TFM_099 stood out for their potential biotechnological relevance, and a comparative analysis of 16S rRNA gene sequences indicated that these strains represented a new species of the genus Hydrotalea. In addition, from the public genomic databases, metagenome-assembled genomes (MAGs) and single-cell amplified genomes (SAGs) could be retrieved that affiliated with this genus. These MAGs and SAGs had been obtained from different environmental samples, such as acid mine drainage or marine sediments. In addition to the description of the new species, the ecological relevance of the members of this genus was demonstrated by means of denitrification, CRISPR-Cas system diversity and heavy metal resistance, as well as their wide geographical distribution and environmental versatility. Supported by the taxonomic study, together with physiological and morphological differences and ecological features, we concluded that strain TMF_100T represented a novel species within the genus Hydrotalea, for which we propose the name Hydrotalea lipotrueae sp. nov.

Description of three new Alteromonas species Alteromonas antoniana sp. nov., Alteromonas lipotrueae sp. nov. and Alteromonas lipotrueiana sp. nov. isolated from marine environments, and proposal for reclassification of the genus Salinimonas as Alteromonas.

In the course of a bioprospective study of marine prokaryotes for cosmetic purposes, four strains, MD_567T, MD_652T, MD_674 and PS_109T, were isolated that 16S rRNA gene affiliation indicated could represent three new species within the family Alteromonadaceae. A thorough phylogenetic, genomic and phenotypic taxonomic study confirmed that the isolates could be classified as three new taxa for which we propose the names Alteromonas antoniana sp. nov., Alteromonas lipotrueae sp. nov. and Alteromonas lipotrueiana sp. nov. In addition, the consistent monophyletic nature of the members of the genera Alteromonas and Salinimonas showed that both taxa should be unified, and therefore we also propose the reclassification of the genus Salinimonas within Alteromonas, as well as new combinations for the species of the former. As the specific epithets profundi and sediminis are already used for Alteromonas species, we created the nomina nova "Alteromonas alteriprofundi" nom. nov. and Alteromonas alterisediminis nom. nov. to accommodate the new names for "Salinimonas profundi" and Salinimonas sediminis. Whole genome comparisons also allowed us to detect the unexpected codification of aromatic hydrocarbon biodegradative compounds, such as benzoate and catechol, whose activity was then demonstrated phenotypically. Finally, the high genomic identity between the type strains of Alteromonas stellipolaris and Alteromonas addita indicated that the latter is a junior heterotypic synonym of Alteromonas stellipolaris.

Distinct ecotypes within a natural haloarchaeal population enable adaptation to changing environmental conditions without causing population sweeps.

Microbial communities thriving in hypersaline brines of solar salterns are highly resistant and resilient to environmental changes, and salinity is a major factor that deterministically influences community structure. Here, we demonstrate that this resilience occurs even after rapid osmotic shocks caused by a threefold change in salinity (a reduction from 34 to 12% salts) leading to massive amounts of archaeal cell lysis. Specifically, our temporal metagenomic datasets identified two co-occurring ecotypes within the most dominant archaeal population of the brines Haloquadratum walsbyi that exhibited different salt concentration preferences. The dominant ecotype was generally more abundant and occurred in high-salt conditions (34%); the low abundance ecotype always co-occurred but was enriched at salinities around 20% or lower and carried unique gene content related to solute transport and gene regulation. Despite their apparent distinct ecological preferences, the ecotypes did not outcompete each other presumably due to weak functional differentiation between them. Further, the osmotic shock selected for a temporal increase in taxonomic and functional diversity at both the Hqr. walsbyi population and whole-community levels supporting the specialization-disturbance hypothesis, that is, the expectation that disturbance favors generalists. Altogether, our results provide new insights into how intraspecies diversity is maintained in light of substantial gene-content differences and major environmental perturbations.

Taxonomic study of nine new Winogradskyella species occurring in the shallow waters of Helgoland Roads, North Sea. Proposal of Winogradskyella schleiferi sp. nov., Winogradskyella costae sp. nov., Winogradskyella helgolandensis sp. nov., Winogradskyella vidalii sp. nov., Winogradskyella forsetii sp. nov., Winogradskyella ludwigii sp. nov., Winogradskyella ursingii sp. nov., Winogradskyella wichelsiae sp. nov., and Candidatus "Winogradskyella atlantica" sp. nov.

Evaluation of bacterial succession with cultivation-dependent strategies during a spring phytoplankton bloom in the North Sea led to the isolation of 41 strains that affiliated with the genus Winogradskyella. Fifteen of the strains were selected for a taxonomic study after discarding clonal cultures. A thorough phylogenetic, genomic and phenotypic analysis of the isolates indicated that they represented eight new species that coexisted in North Sea waters. Molecular data revealed the existence of an as yet uncultivated novel species recurrently binned from the North Sea metagenomes. The metagenome-assembled genomes (MAGs) of this new Winogradskyella were used to classify it as a new Candidatus species. This study represented a new example of the use of the tandem approach of whole cell mass spectrometry linked to 16S rRNA gene sequencing in order to facilitate the discovery of new taxa by high-throughput cultivation, which increases the probability of finding more than a single isolate for new species. In addition, we demonstrated the reasons for classifying MAGs representing recurrently retrieved heterotrophic species that evade cultivation even after an important high-throughput effort. The taxonomic study resulted in the classification of eight new species and one new Candidatus species of the genus Winogradskyella for which we propose the names W. schleiferi sp. nov., W. costae sp. nov., W. helgolandensis sp. nov., W. vidalii sp. nov., W. forsetii sp. nov., W. ludwigii sp. nov., W. ursingii sp. nov., W. wichelsiae sp. nov., and Candidatus "W. atlantica" sp. nov.

Predominance of deterministic microbial community dynamics in salterns exposed to different light intensities.

While the dynamics of microbial community assembly driven by environmental perturbations have been extensively studied, our understanding is far from complete, particularly for light-induced perturbations. Extremely halophilic communities thriving in coastal solar salterns are mainly influenced by two environmental factors-salt concentrations and high sunlight irradiation. By experimentally manipulating light intensity through the application of shading, we showed that light acts as a deterministic factor that ultimately drives the establishment of recurrent microbial communities under near-saturation salt concentrations. In particular, the stable and highly change-resistant communities that established under high-light intensities were dominated (>90% of metagenomic reads) by Haloquadratum spp. and Salinibacter spp. On the other hand, under 37-fold lower light intensity, different, less stable and change-resistant communities were established, mainly dominated by yet unclassified haloarchaea and relatively diverse photosynthetic microorganisms. These communities harboured, in general, much lower carotenoid pigment content than their high-irradiation counterparts. Both assemblage types appeared to be highly resilient, re-establishing when favourable conditions returned after perturbation (i.e. high-irradiation for the former communities and low-irradiation for the latter ones). Overall, our results revealed that stochastic processes were of limited significance to explain these patterns.

Genomic comparison between members of the Salinibacteraceae family, and description of a new species of Salinibacter (Salinibacter altiplanensis sp. nov.) isolated from high altitude hypersaline environments of the Argentinian Altiplano.

The application of tandem MALDI-TOF MS screening with 16S rRNA gene sequencing of selected isolates has been demonstrated to be an excellent approach for retrieving novelty from large-scale culturing. The application of such methodologies in different hypersaline samples allowed the isolation of the culture-recalcitrant Salinibacter ruber second phylotype (EHB-2) for the first time, as well as a new species recently isolated from the Argentinian Altiplano hypersaline lakes. In this study, the genome sequences of the different species of the phylum Rhodothermaeota were compared and the genetic repertoire along the evolutionary gradient was analyzed together with each intraspecific variability. Altogether, the results indicated an open pan-genome for the family Salinibacteraceae, as well as the codification of relevant traits such as diverse rhodopsin genes, CRISPR-Cas systems and spacers, and one T6SS secretion system that could give ecological advantages to an EHB-2 isolate. For the new Salinibacter species, we propose the name Salinibacter altiplanensis sp. nov. (the designated type strain is AN15T=CECT 9105T=IBRC-M 11031T).

The low diverse gastric microbiome of the jellyfish Cotylorhiza tuberculata is dominated by four novel taxa.

Cotylorhiza tuberculata is an important scyphozoan jellyfish producing population blooms in the Mediterranean probably due to pelagic ecosystem's decay. Its gastric cavity can serve as a simple model of microbial-animal digestive associations, yet poorly characterized. Using state-of-the-art metagenomic population binning and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH), we show that only four novel clonal phylotypes were consistently associated with multiple jellyfish adults. Two affiliated close to Spiroplasma and Mycoplasma genera, one to chlamydial 'Candidatus Syngnamydia', and one to bacteroidetal Tenacibaculum, and were at least one order of magnitude more abundant than any other bacteria detected. Metabolic modelling predicted an aerobic heterotrophic lifestyle for the chlamydia, which were found intracellularly in Onychodromopsis-like ciliates. The Spiroplasma-like organism was predicted to be an anaerobic fermenter associated to some jellyfish cells, whereas the Tenacibaculum-like as free-living aerobic heterotroph, densely colonizing the mesogleal axis inside the gastric filaments. The association between the jellyfish and its reduced microbiome was close and temporally stable, and possibly related to food digestion and protection from pathogens. Based on the genomic and microscopic data, we propose three candidate taxa: 'Candidatus Syngnamydia medusae', 'Candidatus Medusoplasma mediterranei' and 'Candidatus Tenacibaculum medusae'.

Moderate halophilic bacteria colonizing the phylloplane of halophytes of the subfamily Salicornioideae (Amaranthaceae).

Halophytes accumulate large amounts of salt in their tissues, and thus are susceptible to colonization by halotolerant and halophilic microorganisms that might be relevant for the growth and development of the plant. Here, the study of 814 cultured strains and 14,189 sequences obtained by 454 pyrosequencing were combined in order to evaluate the presence, abundance and diversity of halophilic, endophytic and epiphytic microorganisms in the phytosphere of leaves of members of the subfamily Salicornioideae from five locations in Spain and Chile. Cultures were screened by the tandem approach of MALDI-TOF/MS and 16S rRNA gene sequencing. In addition, differential centrifugation was used to enrich endophytes for further DNA isolation, 16S rRNA gene amplification and 454 pyrosequencing. Culturable and non-culturable data showed strong agreement with a predominance of Proteobacteria, Firmicutes and Actinobacteria. The most abundant isolates corresponded to close relatives of the species Chromohalobacter canadensis and Salinicola halophilus that comprised nearly 60% of all isolates and were present in all plants. Up to 66% of the diversity retrieved by pyrosequencing could be brought into pure cultures and the community structures were highly dependent on the compartment where the microorganisms thrived (plant surface or internal tissues).

Prokaryotic microbiota in the digestive cavity of the jellyfish Cotylorhiza tuberculata.

The microbiota associated to the gastric cavity of four exemplars of the jellyfish Cotylorhiza tuberculata has been studied by means of cultured-dependent and -independent methods. The pyrosequencing approach rendered a very reduced diversity of Bacteria with four major groups shared by the four exemplars that made up to 95% of the total diversity. The culturing approach recovered low abundant organisms and some of them also detected by the pyrosequencing approach. The major key organisms were related to the genera Spiroplasma, Thalassospira, Tenacibaculum (from the pyrosequencing data), and Vibrio (from the cultivable fraction). Altogether the results indicate that C. tuberculata harbors an associated microbiota of very reduced diversity. On the other hand, some of the major key players may be potential pathogens and the host may serve as dispersal mechanism.

High metabolomic microdiversity within co-occurring isolates of the extremely halophilic bacterium Salinibacter ruber.

Salinibacter ruber is an extremely halophilic member of the Bacteroidetes that thrives in crystallizer ponds worldwide. Here, we have analyzed two sets of 22 and 35 co-occurring S. ruber strains, newly isolated respectively, from 100 microliters water samples from crystalizer ponds in Santa Pola and Mallorca, located in coastal and inland Mediterranean Spain and 350 km apart from each other. A set of old strains isolated from the same setting were included in the analysis. Genomic and taxonomy relatedness of the strains were analyzed by means of PFGE and MALDI-TOF, respectively, while their metabolomic potential was explored with high resolution ion cyclotron resonance Fourier transform mass spectrometry (ICR-FT/MS). Overall our results show a phylogenetically very homogeneous species expressing a very diverse metabolomic pool. The combination of MALDI-TOF and PFGE provides, for the newly isolated strains, the same scenario presented by the previous studies of intra-specific diversity of S. ruber using a more restricted number of strains: the species seems to be very homogeneous at the ribosomal level while the genomic diversity encountered was rather high since no identical genome patterns could be retrieved from each of the samples. The high analytical mass resolution of ICR-FT/MS enabled the description of thousands of putative metabolites from which to date only few can be annotated in databases. Some metabolomic differences, mainly related to lipid metabolism and antibiotic-related compounds, provided enough specificity to delineate different clusters within the co-occurring strains. In addition, metabolomic differences were found between old and new strains isolated from the same ponds that could be related to extended exposure to laboratory conditions.

Description of Bacillus toyonensis sp. nov., a novel species of the Bacillus cereus group, and pairwise genome comparisons of the species of the group by means of ANI calculations.

Strain BCT-7112(T) was isolated in 1966 in Japan from a survey designed to obtain naturally occurring microorganisms as pure cultures in the laboratory for use as probiotics in animal nutrition. This strain, which was primarily identified as Bacillus cereus var toyoi, has been in use for more than 30 years as the active ingredient of the preparation TOYOCERIN(®), an additive for use in animal nutrition (e.g. swine, poultry, cattle, rabbits and aquaculture). Despite the fact that the strain was initially classified as B. cereus, it showed significant genomic differences from the type strains of the B. cereus group that were large enough (ANI values below 92%) to allow it to be considered as a different species within the group. The polyphasic taxonomic study presented here provides sufficient discriminative parameters to classify BCT-7112(T) as a new species for which the name Bacillus toyonensis sp. nov. is proposed, with BCT-7112(T) (=CECT 876(T); =NCIMB 14858(T)) being designated as the type strain. In addition, a pairwise comparison between the available genomes of the whole B. cereus group by means of average nucleotide identity (ANI) calculations indicated that besides the eight classified species (including B. toyonensis), additional genomospecies could be detected, and most of them also had ANI values below 94%. ANI values were on the borderline of a species definition only in the cases of representatives of B. cereus versus B. thuringiensis, and B. mycoides and B. weihenstephanensis.

Evaluation of matrix-assisted laser desorption ionization-time of flight whole cell profiles for assessing the cultivable diversity of aerobic and moderately halophilic prokaryotes thriving in solar saltern sediments.

The moderately halophilic, cultivable fraction of prokaryotes thriving in hypersaline sediments of a solar saltern in Mallorca, Spain, has been studied by means of different cultivation media. A set of 374 isolates retrieved with six different culture conditions was screened, using whole-cell MALDI-TOF MS analysis to classify them into 25 phenotypic clusters at 52% similarity. The phylogenetic inference, made from comparative sequence analyses of the 16S rRNA genes of selected strains, indicated that each phenotypic cluster was comprised of a genealogically homogeneous set of strains. DNA-DNA hybridization (DDH) results among selected strains confirmed that each MALDI-TOF cluster encompassed members of the same species. On the other hand, the intra-cluster diversity, measured by several RAPD (Random Amplified Polymorphic DNA) amplifications, indicated that the clusters corresponded to several populations of the same phylogenetic unit coexisting in the same environment. The results encourage the use of MALDI-TOF MS for further exhaustive studies of the cultivable diversity of hypersaline environments.

Breoghania corrubedonensis gen. nov. sp. nov., a novel alphaproteobacterium isolated from a Galician beach (NW Spain) after the Prestige fuel oil spill, and emended description of the family Cohaesibacteraceae and the species Cohaesibacter gelatinilyticus.

A Gram-negative bacterium designated UBF-P1(T) was isolated from an enrichment culture established in nutrient supplemented artificial sea water with pyrene as a carbon source, and inoculated with a marine fuel oil-degrading consortium obtained from a sand sample collected from the beach of Corrubedo (A Coruña, Galicia, Spain) after the Prestige accidental oil spill. Phylogenetic analysis based on the almost complete 16S rRNA gene sequence affiliated strain UBF-P1(T) with the family Cohaesibacteraceae, Cohaesibacter gelatinilyticus (DSM 18289(T)) being the closest relative species with 92% sequence similarity. Cells were irregular rods, motile, strictly aerobic, catalase and oxidase positive. Ubiquinone 10 was the major respiratory lipoquinone. The major polar lipids comprised diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylmonomethylethanolamine (PME), and phosphatidylcholine (PC). The major fatty acids detected were C(18:1)ω7c, C(19:0) cycloω8c, and C(16:0). The G+C content of strain UBF-P1(T) was 63.9 mol%. The taxonomic comparison with the closest relative based on genotypic, phenotypic and chemotaxonomic characteristics supported that strain UBF-P1(T) could be classified as a novel genus and species, for which the name Breoghania corrubedonensis gen. nov., sp. nov. is proposed. The type strain of this new taxon is UBF-P1(T) (CECT 7622, LMG 25482, DSM 23382).

Lactobacillus bobalius sp. nov., a lactic acid bacterium isolated from Spanish Bobal grape must.

A Lactobacillus strain, designated 203(T), previously isolated from Bobal grape must was characterized phylogenetically, genotypically and phenotypically in order to establish whether it represents a novel species. On the basis of the 16S rRNA gene sequence, strain 203(T) was shown to belong to the genus Lactobacillus, falling within the Lactobacillus alimentarius-Lactobacillus farciminis group and being closely related to the type strains of L. alimentarius, Lactobacillus kimchii and Lactobacillus paralimentarius. DNA-DNA hybridization results confirmed the separate status of strain 203(T) at the species level. To establish the similarities and differences between 203(T) and the three aforementioned closest species, the following methods were used: amplified rDNA restriction analysis, analysis of the 16S-23S rDNA intergenic spacer region, random amplification of polymorphic DNA (RAPD) profiling, ribotyping, carbohydrate fermentation and physiological tests. Strain 203(T) could be differentiated genetically using RAPD analysis and ribotyping. Phenotypically, it can be distinguished from its closest relatives by its ability to grow at pH 3.3, by gas production from gluconate and by certain carbohydrate fermentations. On the basis of these data, strain 203(T) represents a novel species of the genus Lactobacillus, for which the name Lactobacillus bobalius sp. nov. is proposed. The type strain is 203(T) (=CECT 7310(T) =DSM 19674(T)).

Reclassification of Rhodobium marinum and Rhodobium pfennigii as Afifella marina gen. nov. comb. nov. and Afifella pfennigii comb. nov., a new genus of photoheterotrophic Alphaproteobacteria and emended descriptions of Rhodobium, Rhodobium orientis and Rhodobium gokarnense.

The isolation of photoheterotrophic organism C3 from a saline microbial mat led to its taxonomic characterization. Strain C3 could be identified as a member of the species Rhodobium marinum due to the genetic and phenotypic similarities to the type strain of the species (DSM 2698(T)). As a result of a taxonomic study, it was observed that the currently classified species of the genus formed two separate clades, each of them deserving genus status. Rhodobium orientis and Rhodobium gokarnense may be considered as true members of the genus Rhodobium, whereas R. marinum and Rhodobium pfennigii should be reclassified into a new genus. In the light of the genetic and phenotypic evidence observed, we propose that both latter species are reclassified within the new genus Afifella gen. nov., as species Afifella marina comb. nov., and Afifella pfennigii comb. nov., with Af. marina the type species of the genus. In addition, the taxonomic study has revealed that strain DSM 11549, identified as the type strain of the species Rhodopseudomonas julia, may represent a genomovar of Af. marina. The fact that the author of the first classification of R. julia indicates that the strains deposited in the German Collection for Microorganisms (DSM 11549) and American Collection of Type Cultures (ATCC 51105) do not correspond to the original description, makes the authenticity of the strains doubtful. Due to this reason, it is not proposed to reclassify the species.

New method of DNA isolation from two food additives suitable for authentication in polymerase chain reaction assays.

Locust bean gum and guar gum are galactomannans used as additives (E 410 and E 412, respectively) in the food industry as stabilizing agents. Analytical discrimination between the two additives in gums and foods is now feasible by molecular techniques. However, only complex and time-consuming DNA isolation protocols are available to date. We have developed simple improved protocols to obtain enough DNA suitable for PCR amplification from a few milligrams of commercial E 410 and E 412 additives (containing more than 75% polysaccharides). The suspension of additives in water or 10 mM Tris-HCl, pH 8.5, efficiently recovers DNA suitable for authentication in PCR assays. However, the Tris method was much more efficient for the extraction of DNA from E 410 than for E 412 additives. Conversely, the water method was the most suitable for detecting DNA extracted from E 412 or from E 410/E 412 mixtures. Combined with the use of the two specific ribosomal primer pairs previously designed, our methods are well-suited for a fast and simple high-throughput sample treatment of commercial gums for molecular certification.