Search

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 Fără 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. This study was funded by the Spanish Ministry of Science, Innovation and Universities projects PGC2018-096956-B-C41, RTC-2017-6405-1 and PID2021-126114NB-C42, which were also supported by the European Regional Development Fund (FEDER). RRM acknowledges the financial support of the sabbatical stay at Georgia Tech and HelmholzZentrum München by the grants PRX18/00048 and PRX21/00043 respectively also from the Spanish Ministry of Science, Innovation and Universities. This research was carried out within the framework of the activities of the Spanish Government through the “Maria de Maeztu Centre of Excellence” accreditation to IMEDEA (CSIC-UIB) (CEX2021-001198). KTK’s research was supported, in part, by the U.S. National Science Foundation (Award No. 1831582 and No. 2129823). IMG. AC and HLB were financially supported by a grant of the Ministry of Research, Innovation and Digitization, CNCS/CCCDI – UEFISCDI, project number PN-III-P4-ID-PCE-2020-1559, within PNCDI III. HLB acknowledges Ocna Sibiului City Hall (Sibiu County, Romania) for granting the access to Fără Fund Lake and A. Baricz and D.F. Bogdan for technical support during sampling and sample preparation. MBS thanks Dominion Salt for their assistance in sample Lake Grassmere. MELL acknowledges the financial support of the Argentinian National Scientific and Technical Research Council (Grant CONICET-NSFC 2017 N° IF-2018-10102222-APN-GDCT-CONICET) and the National Geographic Society (Grant # NGS 357R-18). BPH was supported by NASA (award 80NSSC18M0027). TV acknowledges the “Margarita Salas” postdoctoral grant, funded by the Spanish Ministry of Universities, within the framework of Recovery, Transformation and Resilience Plan, and funded by the European Union (NextGenerationEU), with the participation of the University of Balearic Islands (UIB).

Two moderately halophilic and psychrotolerant new Mycoplasma species were isolated from common cephalopods. Three strains were isolated in pure culture from two individual European flying squid (Todarodes sagittatus), and two individual octopuses (Octopus vulgaris). The strains showed optimal growth at 25 °C and a salinity of 3% (w/v) NaCl. Molecular analyses revealed that the isolates belonged to two new, but phylogenetically related species, divergent from all previously described Mollicutes, representing the first marine isolates of the class, and also the first Mycoplasma strains for which NaCl requirement has been demonstrated. A genome search against all available marine metagenomes and 16S rRNA gene databases indicated that these two species represent a novel non-free-living marine lineage of Mollicutes, specifically associated with marine animals. Morphology and physiology were compatible with other members of this group, and genomic and phenotypic analyses demonstrated that these organisms represent two novel species of the genus Mycoplasma, for which the names Mycoplasma marinum sp. nov. and Mycoplasma todarodis sp. nov. are proposed; the type strains are PET (DSM 105487T, CIP 111404T) and 5HT (DSM 105,488T, CIP 111405T), respectively., RRM acknowledges the Spanish Ministry of Economy project CLG2015_66686-C3-1-P also supported with European Regional Development Fund (FEDER) funds. ASR acknowledges the Gobierno de Canarias (Spain) project P2007/046. RRM acknowledges the financial support of the sabbatical stay at Georgia Institute of Technology supported by the grant PRX18/00048 of the Ministry of Sciences, Innovation and Universities. KTK’s research was supported, in part, by the U.S. National Science Foundation (Award No. 1,759,831).

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., This study was funded by the Spanish Ministry of Sciences, Innovation and Universities projects PHRYEN_CLG2015_66686-C3-1-P, MARBIOM_RTC-2017-6405-1 and MICROMATES_PGC2018-096956-B-C41, and was also supported by European Regional Development Fund (FEDER) funds. RA was financed by the Max Planck Society. RRM acknowledges the financial support of a sabbatical stay at Georgia Tech supported by the grant PRX18/00048 of the Ministry of Sciences, Innovation and Universities.

Microbial degradation of algal biomass following spring phytoplankton blooms has been characterised as a concerted effort among multiple clades of heterotrophic bacteria. Despite their significance to overall carbon turnover, many of these clades have resisted cultivation. One clade known from 16S rRNA gene sequencing surveys at Helgoland in the North Sea, was formerly identified as belonging to the genus Ulvibacter. This clade rapidly responds to algal blooms, transiently making up as much as 20% of the free-living bacterioplankton. Sequence similarity below 95% between the 16S rRNA genes of described Ulvibacter species and those from Helgoland suggest this is a novel genus. Analysis of 40 metagenome assembled genomes (MAGs) derived from samples collected during spring blooms at Helgoland support this conclusion. These MAGs represent three species, only one of which appears to bloom in response to phytoplankton. MAGs with estimated completeness greater than 90% could only be recovered for this abundant species. Additional, less complete, MAGs belonging to all three species were recovered from a mini-metagenome of cells sorted via flow cytometry using the genus specific ULV995 fluorescent rRNA probe. Metabolic reconstruction indicates this highly abundant species most likely degrades proteins and the polysaccharide laminarin. Fluorescence in situ hybridisation showed coccoid cells, with a mean diameter of 0.78mm, with standard deviation of 0.12μm. Based on the phylogenetic and genomic characteristics of this clade, we propose the novel candidate genus Candidatus Prosiliicoccus, and for the most abundant and well characterised of the three species the name Candidatus Prosiliicoccus vernus.

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). Fil: Viver, Tomeu. Consejo Superior de Investigaciones Cientificas. Instituto Mediterraneo de Estudios Avanzados; España Fil: Orellana, Luis. Instituto Tecnológico de Georgia; Estados Unidos. Georgia Institute of Techology; Estados Unidos Fil: González Torres, Pedro. Universidad de Alicante; España Fil: Díaz, Sara. Consejo Superior de Investigaciones Cientificas. Instituto Mediterraneo de Estudios Avanzados; España Fil: Urdiain, Mercedes. Consejo Superior de Investigaciones Cientificas. Instituto Mediterraneo de Estudios Avanzados; España Fil: Farias, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina Fil: Benes, Vladimir. European Molecular Biology Laboratory Heidelberg; Fil: Kaempfer, Peter. Justus Liebig University Giessen; Fil: Shahinpei, Azadeh. University Of Tehran; Irán Fil: Ali Amoozegar, Mohammad. University Of Tehran; Irán Fil: Amann, Rudolf. Max Planck Institut Für Marine Mikrobiologie; Fil: Antón, Josefa. Universidad de Alicante; España Fil: Konstantinidis, Konstantinos T.. Georgia Institute of Techology; Estados Unidos. Instituto Tecnológico de Georgia; Estados Unidos Fil: Rosselló Móra, Ramon. Consejo Superior de Investigaciones Cientificas. Instituto Mediterraneo de Estudios Avanzados; España

Seawater contains free-living and particle-attached bacteria. Only a small fraction is cultivable on plates. As free-living and particle-associated bacteria differ in their physiological traits, their cultivability on plates may coincide with particle association. Using filtration and Imhoff sedimentation cones, particles were collected during a spring phytoplankton bloom off Helgoland (North Sea) in order to obtain particle-associated bacteria as inocula. Direct dilution plating resulted in 526 strains from 3 µm filtration retentates and 597 strains from settled particles. Motile Gammaproteobacteria from the genera Pseudoalteromonas, Shewanella, Psychrobacter, Vibrio and Colwellia, as well as particle-attached Flavobacteriia affiliating with the genera Tenacibaculum and Gramella, were frequently isolated. As a result, a diverse collection comprised of 266 strains was deposited. Two strains were most likely to represent novel genera and 78 strains were probably novel species. Recently, a high-throughput cultivation study from the same site using seawater as an inoculum had retrieved 271 operational phylogenetic units (OPUs) that represented 88% of the 4136 characterized strains at the species level. A comparison of 16S rRNA gene sequences revealed that the collection obtained matched 104 of the 271 seawater OPUs at the species level and an additional 113 at the genus level. This large overlap indicated a significant contribution of particle-associated bacteria to the cultivable microbiome from seawater. The presence of 49 genera not identified in the larger seawater study suggested that sample fractionation was an efficient strategy to cultivate rare members of the planktonic microbiome. The diverse collection of heterotrophic bacteria retrieved in this study will be a rich source for future studies on the biology of particle-associated bacteria.

Coastal phytoplankton blooms are frequently followed by successive blooms of heterotrophic bacterial clades. The class Flavobacteriia within the Bacteroidetes has been shown to play an important role in the degradation of high molecular weight substrates that become available in the later stages of such blooms. One of the flavobacterial clades repeatedly observed over the course of several years during phytoplankton blooms off the coast of Helgoland, North Sea, is Vis6. This genus-level Glade belongs to the family Cryomorphaceae and has been resistant to cultivation to date. Based on metagenome assembled genomes, comparative 16S rRNA gene sequence analyses and fluorescence in situ hybridization, we here propose a novel candidate genus Abditibacter, comprising three novel species Candidatus Abditibacter vernus, Candidatus Abditibacter forsetii and Candidatus Abditibacter autumni. While the small genomes of the three novel photoheterotrophic species encode highly similar gene repertoires, including genes for degradation of proteins and algal storage polysaccharides such as laminarin, two of them - Ca. A. vernus and Ca. A. forsetii - seem to have a preference for spring blooms, while Ca. A. autumni almost exclusively occurs in late summer and autumn. (C) 2020 The Author(s). Published by Elsevier GmbH.

There is little doubt that we are approaching an era where changes will occur in the traditional way we taxonomically classify microbes; the accumulating genomic, and multi-omic data in general, are simply calling for it! More specifically, an exponentially increasing number of metagenome-assembled genomes (MAGs) and single-cell amplified genomes (SAGs) are becoming available these days, most of which are representatives of yet uncultured species that are important for ecosystem functions, and sometimes disease. Last year alone, more than 3000 MAGs were reported, each of which would represent a new species by genomic standards, vastly outnumbering the ∼1000 newly classified species based on classic taxonomic approaches and isolates. Thus, there is an urgent need to start naming (nomenclature component of taxonomy) and devising standards to classifying (classification component of taxonomy) the important uncultivated Bacteria and Archaea in order to facilitate communication among scientists and the public. For instance, it is not uncommon nowadays that different scientists are working on the same uncultured microorganism(s), but they do not know it in the absence of a supervised and stable taxonomic framework for the uncultured. Furthermore, the alphanumeric names (e.g., SUP05, SAR11) tentatively given to uncultivated taxa are not informative of the phenotype or ecology of the corresponding organisms; neither are these names regulated, creating a chaotic situation that is already difficult to manage. Unfortunately, the existing approach for cataloguing uncultivated taxa, the Candidatus concept, in its current form, is unable to deal with the type and volume of accumulating omic data without substantial changes. The changes would likely include – but not be limited to – the adoption of new, genome-based techniques and standards for classification to complement the traditional techniques that are not well applicable to uncultivated organisms, as well as the acceptance of the DNA sequence as the type material in the Bacteriological Code and the prioritization of taxonomic names for nomenclature in order to further promote the description of uncultivated taxa.

Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) with rRNA-targeted oligonucleotide probes has significantly improved the identification of microorganisms in various environmental samples. However, one of the major constraints of CARD-FISH is the low probe penetration due to the high molecular weight of the horseradish peroxidase (HRP) label. Recently, this limitation has been overcome by a novel signal amplification approach termed in situ DNA-hybridization chain reaction (in situ DNA-HCR). In this study, we present an improved and accelerated in situ DNA-HCR protocol (quickHCR-FISH) with increased signal intensity, which was approximately 2 times higher than that of standard in situ DNA-HCR. In addition, the amplification time was only 15 min for the extension of amplifier probes from the initiator probe compared to 2h in the original protocol. The quickHCR-FISH was successfully tested for the quantification of marine bacteria with low rRNA contents in both seawater and sediment samples. It was possible to detect the same number of marine bacteria with quickHCR-FISH compared to CARD-FISH within only 3h. Thus, this newly developed protocol could be an attractive alternative to CARD-FISH for the detection and visualization of microorganisms in their environmental context.

The 23S rRNA gene was evaluated as target for the development of Sybr Green-based quantitative PCR (qPCR) for the analysis of nitrogen-fixing members of the genus Frankia or subgroups of these in soil. A qPCR with a primer combination targeting all nitrogen-fixing frankiae (clusters 1, 2 and 3) resulted in numbers similar to those obtained with a previously developed qPCR using nifH gene sequences, both with respect to introduced and indigenous Frankia populations. Primer combinations more specifically targeting three subgroups of the Alnus host infection group (cluster 1) or members of the Elaeagnus host infection group (cluster 3) were specific for introduced strains of the target group, with numbers corresponding to those obtained by quantification of nitrogen-fixing frankiae with both the 23S rRNA and nifH genes as target. Method verification on indigenous Frankia populations in soils, i.e. in depth profiles from four sites at an Alnus glutinosa stand, revealed declining numbers in the depth profiles, with similar abundance of all nitrogen-fixing frankiae independent of 23S rRNA or nifH gene targets, and corresponding numbers of one group of frankiae of the Alnus host infection only, with no detections of frankiae representing the Elaeagnus, Casuarina, or a second subgroup of the Alnus host infection groups.

Nitrogen (N 2 ) fixation is a globally important process often mediated by diazotrophic cyanobacteria in the open ocean. In 2010, seawater was collected near Cape Verde to identify and measure N 2 and carbon (C) fixation by unicellular diazotrophic cyanobacteria. The nifH gene abundance (10 4 –10 6 nifH L −1 ) and nifH gene transcript abundance (10 2 –10 4 cDNA nifH L −1 ) for two unicellular groups, UCYN-A and UCYN-B, were detected. UCYN-A was also identified and quantified (10 4 –10 5 cells L −1 ) by new probes (UCYN-A732 and UCYN-A159) using Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization (CARD-FISH) assays. The UCYN-A were observed as free cells or attached to a larger unidentified eukaryotic cell. A Halogen In Situ Hybridization-Secondary Ion Mass Spectrometry (HISH-SIMS) assay using the UCYN-A732 probe was applied on samples previously incubated with 13 C-bicarbonate and 15 N 2 . Free UCYN-A cells were enriched in both 13 C and 15 N and estimated C and N 2 fixation rates for UCYN-A were lower compared to co-occurring unicellular cyanobacteria cells similar in size (3.1–5.6 μm) and pigmentation to diazotroph Crocosphaera watsonii . Here, we identify and quantify two common co-occurring unicellular groups and measure their cellular activities by nanoSIMS.

Members of the phylum Bacteroidetes, which was originally defined as a monophyletic branch encompassing the genera Cytophaga, Flavobacterium and Bacteroides (CFB), are widely studied due to their importance in environmental and gut microbiology. As a consequence, the number of species names with standing in nomenclature has doubled in the past five years. In this study, a revision of an earlier phylogeny of Bacteroidetes has been performed using the 16S rRNA gene as a backbone in combination with the 23S rRNA gene, as well as multilocus sequence analysis (MLSA) of 29 orthologous protein sequences, and indels in the sequences of the beta subunit of the F-type ATPase and the alanyl-tRNA synthetase. In addition, taxonomic data for Bacteroidetes has been updated by considering the orphan species list, signature nucleotides in the 16S rRNA sequence, the list of outlier species, and discrepancies with the current taxonomy at the genus rank level. As a result, seven new taxa are proposed within Bacteroidetes (Chitinophagia classis nov., Chitinophagales ord. nov., Crocinitomicaceae fam. nov., Odoribacteraceae fam. nov., Hymenobacteraceae fam. nov., Thermonemataceae fam. nov. and Persicobacteraceae fam. nov.), as well as one new phylum Rhodothermaeota phyl. nov. that contains two classes, two orders, four families and a new genus with two new combinations., This study was funded by the Max Planck Society, the Spanish Ministry of Economy and Competitivity projects CGL2012-39627-C03-03 and CLG2015-66686-C3-1-P that also supported the work with European Regional Development Fund (FEDER) funds, the preparatory phase of the Microbial Resource Research Infrastructure (MIRRI) funded by the EU (grant number 312251), and the financial support of Deep Blue Sea Enterprise S.L. RMJ would like to thank the Max Planck Institute for Marine Microbiology for funding his PhD program and Elsevier Ltd. in funding the maintenance of the LTP database. RRM acknowledges the financial support of the Spanish Ministry through the project PR2015-00008 for international scientific exchange.

One of the main goals of microbial ecologists is to assess the contribution of distinct bacterial groups to biogeochemical processes, e.g. carbon cycling. Until very recently, it was not possible to quantify the uptake of a given compound at single cell level. The advent of nano-scale secondary-ion mass spectrometry (nanoSIMS), and its combination with halogen in situ hybridization (HISH) opened up this possibility. Despite its power, difficulties in cell identification during analysis of environmental samples might render this approach challenging for certain applications. A pilot study, designed to quantify the incorporation of phytoplankton-derived carbon by the main clades of heterotrophic aquatic bacteria (i.e. Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes), is used to exemplify and suggest potential solutions to these technical difficulties. The results obtained indicate that the main aquatic bacterial clades quantitatively differ in the incorporation of algae-derived organic matter. From the methodological point of view, they highlight the importance of the concentration of the target cells, which needs to be sufficient to allow for a rapid mapping under the nanoSIMS. Moreover, when working with highly productive waters, organic and inorganic particles pose a serious problem for cell recognition based on HISH-SIMS. In this work several technical suggestions are presented to minimize the above mentioned difficulties, including alternatives to improve the halogen labeling of the cells and proposing the use of a combination of FISH and HISH along with a mapping system. This approach considerably enhances the reliability of cell identification and the speed of the subsequent nanoSIMS analysis in such complex samples.

Members of the highly diverse bacterial phylum Verrucomicrobia are globally distributed in various terrestrial and aquatic habitats. They are key players in soils, but little is known about their role in aquatic systems. Here, we report on the design and evaluation of a 16S rRNA-targeted probe set for the identification of Verrucomicrobia and of clades within this phylum. Subsequently, the probe set was applied to a study concerning the seasonal abundance of Verrucomicrobia in waters of the humic lake Grosse Fuchskuhle (Germany) by catalyzed reporter deposition fluorescence in situ hybridization. The lake hosted diverse Verrucomicrobia clades in all seasons. Either Spartobacteria (up to 19%) or Opitutus spp. (up to 7%) dominated the communities, whereas Prosthecobacter spp. were omnipresent in low numbers (

We show that non-denaturing gel electrophoresis, or single-stranded conformational polymorphism (SSCP), can be used to separate mixtures of full-length rRNAs. Individual bands can then be excised for identification by RT-PCR and sequencing. This has the advantage over profiling methods such as DGGE and T-RFLP that no PCR amplification is involved prior to sequencing; thus, extraction biases aside, it should yield a quantitative picture of community composition in terms of ribosome content. To simplify banding patterns, RNA subsamples (e.g. bacterial 16S rRNA) can first be isolated by magnetic bead capture hybridization. Alternatively, oligonucleotide-directed ribonuclease H (RNase H) digestion can be used to identify bands of interest by running digested samples in parallel to undigested ones. We illustrate the use of this technique to identify a potentially predominant species in a hypersaline microbial mat. We anticipate that rRNA-SSCP will be useful for community profiling; for clone library construction by directed cloning of individual rRNAs; and for following incorporation of radiolabeled substrates at the species level, by gel autoradiography, without advance information or guesswork about which species might be active and abundant.

Molecular biological methods were used to investigate the microbial diversity and community structure in intertidal sandy sediments near the island of Sylt (Wadden Sea) at a site which was characterized for transport and mineralization rates in a parallel study (D. de Beer, F. Wenzhöfer, T. Ferdelman, S.E. Boehme, M. Huettel, J.E.E. van Beusekom, M.E. Böttcher, N. Musat, N. Dubilier, Transport and mineralization rates in North Sea sandy intertidal sediments, Sylt-Romo Basin, Wadden Sea, Limnol. Oceanogr. 50 (2005) 113-127). Comparative 16S rRNA sequence analysis revealed a high bacterial diversity. Most sequences retrieved by PCR with a general bacterial primer set were affiliated with Bacteroidetes, Gammaproteobacteria, Deltaproteobacteria and the Pirellula cluster of Planctomycetales. Fluorescence in situ hybridization (FISH) and slot-blot hybridization with group-specific rRNA-targeted oligonucleotide probes were used to characterize the microbial community structure over depth (0-12 cm) and seasons (March, July, October). We found high abundances of bacteria with total cell numbers up to 3 x 10(9) cells ml(-1) and a clear seasonal variation, with higher values in July and October versus March. The microbial community was dominated by members of the Planctomycetes, the Cytophaga/Flavobacterium group, Gammaproteobacteria, and bacteria of the Desulfosarcina/Desulfococcus group. The high abundance (1.5 x 10(7)-1.8 x 10(8) cells ml(-1) accounting for 3-19% of all cells) of presumably aerobic heterotrophic polymer-degrading planctomycetes is in line with the high permeability, deep oxygen penetration, and the high rates of aerobic mineralization of algal biomass measured in the sandy sediments by de Beer et al. (2005). The high and stable abundance of members of the Desulfosarcina/Desulfococcus group, both over depth and season, suggests that these bacteria may play a more important role than previously assumed based on low sulfate reduction rates in parallel cores (de Beer et al., 2005).

A microbial mat from the Black Sea shelf was analyzed by a metagenomic approach. While the habitat and its microbial community are characterized by anaerobic methane oxidation, a 79 kb contiguous DNA sequence obtained from the same mat provided first evidence for the concomitant presence of the capacity for anaerobic benzoate degradation. Benzoyl-CoA is one central intermediate of anaerobic aromatic degradation, among others. Within a stretch of 31 kb, all genes required for the complete pathway of anaerobic benzoate degradation (catabolic island) were identified, including the four subunits of the key enzyme benzoyl-CoA reductase (bcrCBAD), which catalyzes the ATP-driven 2-electron reduction of the aromatic ring. Genes for a ketoacid:acceptor oxidoreductase (korABC) and a ferredoxin (fdx), which are required for generation of a suitable electron donor, were also detected. The majority of the identified catabolic gene products are most similar to their respective orthologs from the denitrifying freshwater bacterium Azoarcus evansii, and the genes are also similarly organized. Due to the lack of established markers, the phylogenetic affiliation of the source organism remains unclear. The presented findings indicate that the metabolic diversity of the Black Sea mat is wider than currently known and that probably other bacteria than those of the methane-oxidizing consortia contribute to aromatic degradation in this anoxic habitat.

Summary Almost complete 23S rRNA gene sequences were obtained from 11 Alphaproteobacteria isolated from marine surface water of the German Bight. Five of the strains belong to the “marine alpha” group, a phylogenetic cluster which encompasses members of the genus Roseobacter and closely related bacteria. Phylogenetic sequence analysis based on 52 published as well as unpublished complete 23S rDNA sequences from Alphaproteobacteria including the newly obtained was in general consistent with the 16S rRNA gene sequence-derived phylogeny. 16S and 23S rRNA based phylogenies both showed a distinct cluster for strains associated with the “marine alpha” group. The suitability of both markers for the design of oligonucleotide probes targeting selected groups of Alphaproteobacteria was systematically evaluated and compared in silico . Six clusters of sequences covering different phylogenetic levels as well as two strains were selected in a case study. To compensate for the quantitative difference in the two data sets, the 16S rRNA dataset was truncated to sequences with an equivalent in the 23S rRNA data set. Our results show, that the overall number of phylogenetically redundant probes available could be more than doubled by extending probe design to the 23S rRNA. For small clusters of high sequence similarity and single strains, up to 8 times more discriminating binding sites were provided by the 23S rRNA.

Oligonucleotide probes labeled with fluorescent dyes are used in a variety of in situ applications to detect specific DNA or RNA molecules. It has been described that probe fluorescence might be quenched upon hybridization in a sequence specific way. Here, a set of 17 oligonuleotides labeled with 6-carboxyfluorescein was used to examine the relevance of nucleotide specific quenching for fluorescence in situ hybridization (FISH) to whole fixed bacterial cells. Probes quenched upon hybridization to a guanine-rich region of purified RNA in solution were not quenched upon FISH. Among other factors the high protein concentration within cells may prevent quenching of probe fluorescence in situ.

An aggregate-attached bacterium, strain 88/2-7, was isolated from samples of the Southern Ocean and investigated in a polyphasic approach. The novel marine isolate is an aerobic, Gram-negative, oxidase- and catalase-positive, non-motile short rod and grows in form of cream-colored colonies. Growth was observed at 5-35 degrees C. The bacterium tolerated concentrations of 0-13% (w/v) NaCl and utilized a relatively restricted spectrum of carbon sources. The analysis of the fatty acids revealed 18:1 cis 9 (18:1omega9c) as main fatty acid. The G+C content of the DNA was approximately 42 mol%. The sequence of the 16S rDNA assigned strain 88/2-7 to the gamma-subclass of Proteobacteria with a similarity of 99.65% to Psychrobacter proteolyticus (DSM 13887T). A DNA-DNA-hybridization study showed only 26.8% renaturation to the respective strain. Based on the morphological, physiological and molecular properties of the new isolate, the name Psychrobacter nivimaris sp. nov. (type strain 88/2-7T) is proposed.

Summary Bacteria of the Cytophaga-Flavobacterium-Bacteroides phylum (CFB-phylum) are numerically important members of many microbial communities. A suite of five 16S rRNA-targeted oligonucleotide probes for members of this group is described which was designed to dominantly target bacteria of the CFB-phylum that are found in particular habitats. For this we initially performed a literature survey for the sources and sites of isolation of hitherto described members of the CFB-phylum. Probe CFB286 is mostly complementary to the 16S rRNA of species originally isolated from freshwater habitats, however, detects some marine and soil isolates and is the only probe which includes some food isolates. Probe CFB563 detects marine as well as animal-associated isolates. Probe CFB719, which also detects some environmental isolates, and probe CFB972 are mostly targeting animal-associated isolates. All probes are complementary to a variety of human-associated species within the CFB-phylum which, in the data set investigated (October 1998), made up 46% of all 16S rRNA sequences from the CFB-phylum. We conclude that it is difficult to find habitat-specific probes for members of the CFB-phylum and that the design of probes for monophyletic groups should remain the standard approach. Applicability of the probes for fluorescence in situ hybridization and specificity for single cell detection were evaluated for the four mentioned probes whereas the fifth, probe CFB1082, which almost exclusively targets human-associated species, was not further characterized. The new probes are of limited determinative value and should be used together with the already established probes for the CFB-phylum. It is the hybridization pattern observed for a given cell or culture with the enlarged probe set that is suggestive for its affiliation with a defined group within the CFB-phylum.

In situ hybridization with rRNA-targeted oligonucleotide probes has become a widely applied tool for direct analysis of microbial population structures of complex natural and engineered systems. In such studies probe EUB338 (AMANN et al., 1990) is routinely used to quantify members of the domain Bacteria with a sufficiently high cellular ribosome content. Recent reevaluations of probe EUB338 coverage based on all publicly available 16S rRNA sequences, however, indicated that important bacterial phyla, most notably the Planctomycetales and Verrucomicrobia, are missed by this probe. We therefore designed and evaluated two supplementary versions (EUB338-II and EUB338-III) of probe EUB338 for in situ detection of most of those phyla not detected with probe EUB338. In situ dissociation curves with target and non-target organisms were recorded under increasing stringency to optimize hybridization conditions. For that purpose a digital image software routine was developed. In situ hybridization of a complex biofilm community with the three EUB338 probes demonstrated the presence of significant numbers of probe EUB338-II and EUB338-III target organisms. The application of EUB338, EUB338-II and EUB338-III should allow a more accurate quantification of members of the domain Bacteria in future molecular ecological studies.

Fluorescence in situ hybridization of activated sludge samples from a municipal wastewater treatment plant using oligonucleotide probes specific for Acidovorax demonstrated that these bacteria are highly abundant in this environment. For the targeted cultivation of representatives belonging to this genus, isolates grown on agar plates after serial dilution were screened by whole-cell hybridization with specific probes. The obtained strains clustered in two phylogenetic groups as determined by 16S rRNA gene sequence analyses. The isolates of one cluster were phylogenetically and genotypically closely related to A. delafieldii. In contrast, the strains of the other cluster were genotypically and phenotypically distinct from the hitherto known Acidovorax species. Therefore, a new species, Acidovorax defluvii sp. nov., was proposed for these strains. The main characteristics of the newly defined species are as follows: Gram-negative, motile or non-motile rods with rounded ends, often with large polyhydroxybutyrate granules. In broth cultures flocs are formed. Test for cytochrome oxidase is positive with all strains. The majority of strains is catalase positive and reduces nitrate. All strains are metabolically inactive against most carbohydrates and organic acids. Fatty acid patterns are typical for the genus Acidovorax. The guanine-plus-cytosine content of DNAs varies between 62 and 64 mol%. The type strain of A. defluvii is BSB411T (DSM 12644). A new 16S rRNA-targeted oligonucleotide probe reacting by in situ hybridization with all known Acidovorax species, including A. defluvii sp. nov., was designed.

Summary Achromatium oxaliferum was first described in 1893 by Schewiakoff as an unusually large bacterium living in freshwater sediments. Up to now no pure culture is available. Physical enrichments of achromatia collected from the acidic Lake Fuchskuhle, which houses a peculiar, smaller variety, and the neutral Lake Stechlin were investigated by the cultivation-independent rRNA approach. PCR in combination with cloning and sequencing was used for the retrieval of 24 partial and 4 nearly full-length 16S rRNA sequences that formed two distinct phylogenetic clusters. Fluorescence-in-situ-hybridization (FISH) with four 16S rRNA-targeted oligonucleotide probes unambiguously assigned the different sequences to either regular, large A. oxaliferum cells or to the smaller Lake Fuchskuhle population, tentatively named “A. minus” The two Achromatium sp. 16S rRNA sequence clusters form a stable deep branch in the gamma subclass of the class Proteobacteria. The closest cultivated relatives are Chromatium vinosum, Rhabdochromatium marinum and Ectothiorhodospira halophila with 16S rRNA similarities of 86.2 to 90.5%. Profound differences in the population structure of achromatia were revealed in the two lakes by FISH. In one sample from Lake Stechlin three genotypes could be visualized, and 49% of the cells were assigned to A. oxaliferum clone AST01, 28% to Achromatium sp. genotype AFK192/AFK433 and 23% to Achromatium sp. genotype AFK192/AST433. In contrast, a morphologically and phylogenetically homogeneous population of “A. minus”, was present in Lake Fuchskuhle.

Summary A method is described for the in situ detection of individual whole fixed cells of Escherichia coli containing ColE1-related plasmids. It makes use of fluorescence in situ hybridization (FISH) and the regulatory RNA II as a target molecule for both, Cy3- and HRP-labeled olinucleotide probes. Various methods for signal amplification were compared. Probes targeting the regulatory RNA I did not result in the in situ detection of plasmid-bearing cells.

Presentación del número de la revista editado por estos tres autores

Broad-scale differences in soil microbial community composition were analyzed in two contrasting soils using DNA reassociation and % G + C profiles for analysis on the community-level, and filter- and whole cell hybridization techniques for a coarse-level characterization of larger phylogenetic groups of bacteria. Reassociation analysis of DNA from bacterial fractions extracted from the organic soil Seim and the mineral soil Hau revealed similar complexity of the communities with 5700 and 4900 different bacterial genomes (g soil [dry wt])-1, respectively. Thermal denaturation studies showed wide % G + C distributions in DNA from bacteria of both soils. Differences in the median % G + C with 55 to 61% for the bacterial community in soil Seim and 61 to 66% for that in soil Hau indicated a higher proportion of bacteria with a high DNA G + C content in soil Hau. In situ hybridization with fluorescent (Cy3-labeled) probes targeting larger phylogenetic groups showed minor differences between both soils, and between direct detection of bacteria in dispersed soil slurries and in bacterial fractions extracted from soils through about 90% of the total bacteria were lost during extraction. In dispersed slurries of both soils, only probes ALF1b, SRB385, and PLA46 hybridized to cells accounting for more than 1% of the DAPI-stained cells, while numbers obtained after hybridization with probes ARCH915, BET42a, GAM42a, HGC69a, and CF319a were below the detection limit set at1%. These results were confirmed by in situ hybridization with horseradish peroxidase (HRP)-labeled probes and subsequent Cy3-tyramide signal amplification. In contrast, dot blot hybridization with probe HGC69a indicated significant amounts of Gram-positive bacteria with a high DNA G + C content in both soils. These could subsequently be visualized in non-dispersed soil slurries by in situ hybridization with HRP-labeled probe HGC69a and Cy3-tyramide signal amplification. Filamentous Gram-positive bacteria with a high DNA G + C content, likely actinomycetes, which are present in soil Hau in significant numbers are obviously destroyed by procedures used for soil dispersion.

Summary Identification of enterococci species by hybridization with recently designed species-specific and groupspecific 23S rDNA-targeted oligonucleotide probes was superior to results obtained with a common biochemical test panel. Considering these findings, a molecular biological procedure for the detection of enterococci in water samples was developed. A short enrichment is followed by an amplification step and a hybridization reaction in microtiter plate format. The detection limit is about 1 CFU/ml, and results are available within 26 h.