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Denitrification is an alternative type of anaerobic respiration in which nitrate is reduced to gaseous products via nitrite. The key step in this process is the reduction of nitrite to nitric oxide, which is catalyzed by two structurally different but functionally equivalent forms of nitrite reductase encoded by thenirKandnirSgenes. Cultivation-independent studies based on these functional marker genes showed that in the environment there was a dominance of organisms withnirKandnirSgenes presumably derived from organisms that have not been cultured yet. However, the phylogenetic affiliation of these organisms has not been resolved since the ability to denitrify is widespread in phylogenetically unrelated organisms. To unravel the phylogeny of the organisms from which the nitrite reductase (nirK) genes originated, one option is to use a special variant of whole-cell hybridization termed recognition of individual genes-fluorescence in situ hybridization (RING-FISH). In RING-FISH a multiply labeled transcript polynucleotide probe is used to detect a single gene on the bacterial chromosome during FISH. Here, RING-FISH was used with laboratory cultures and environmental samples, such as activated sludge. Furthermore, probe-based cell sorting using magnetic beads could also be carried out with mixtures of pure cultures, which led to effective depletion of thenirK-negative organism but capture of thenirK-positive organism, which was demonstrated by terminal restriction fragment length polymorphism analysis based on 16S rRNA genes. The results indicate that RING-FISH coupled with probe-based cell sorting could be used with environmental samples, which could provide a means for phylogenetic classification ofnirK-type denitrifiers. Thus, the results of RING-FISH could increase our understanding of the phylogeny and function of denitrifying microorganisms in the environment.

Escherichia coli K-12, B, C and W strains and their derivates are declared in biological safety guidelines as risk group 1 organisms as they are unable to colonise the human gut. Differentiation and identification of these safety strains is mainly based on pulsed-field gel electrophoresis (PFGE), phage sensitivity tests or PCR-based methods. However, these methods are either tedious and time consuming (phage sensitivity, PFGE) or based on single specific fragments (PCR) or patterns (PFGE) lacking additional information for further differentiation of the strains. In the current study, subtractive hybridisation techniques were applied to detect specific DNA fragments which were used to design a microarray (chip) for accurate and simple identification of these organisms, and to differentiate them from other E. coli strains. The chip can be used to identify E. coli safety strains and monitor them during ongoing experiments for changes in their genome and culture purity. The hybridisation layout of the microarray was arranged in such a way that the respective lineages of safety strains could be easily identified as distinct letters (K, B, C or W). Differentiation of single strains or subtyping was possible with further probes. In addition, a set of probes targeting genes coding for common virulence factors has been included, both to differentiate safety strains from pathogenic variants and to make sure that no transfer of these genes happens during handling or storage. The reliability of the approach has been tested on a comprehensive selection of E. coli laboratory strains and pathogenic representatives.

Bacterial endosymbionts of protozoa were often described as new species by protozoologists mainly on the basis of few morphological characters and partly by host specificity. Many of these species have never been validated by prokaryotic microbiologists whose taxonomic rules are quite different from those of protozoologists, who use the Zoological Code of Nomenclature. “Caedibacter macronucleorum”Fokin and Gortz 1993, an endosymbiont of Paramecium duboscqui, belongs to this category. Here we provide the molecular characterization of this organism and of its host P. duboscqui strain Ku4-8. Bacterial 16S rRNA gene sequence analysis proved that “C. macronucleorum” belongs to the Alphaproteobacteria. It is closely related to Caedibacter caryophilus but not to Caedibacter taeniospiralis, which belongs to the Gammaproteobacteria. “Caedibacter macronucleorum” and C. caryophilus 16S rRNA genes show a similarity value of 99%. This high 16S rRNA sequence similarity and the lack of a specific oligonucleotide probe for distinguishing the two endosymbionts do not allow validating “C. macronucleorum” as a provisional taxon (Candidatus). Nevertheless, “C. macronucleorum” and C. caryophilus can be easily discriminated on the basis of a highly variable stretch of nucleotides that interrupts the 16S rRNA genes of both organisms.

Refractile inclusion bodies, called R-bodies were observed within the cells of some bacterial strains. They are protein ribbons, which are typically coiled into cylindrical structures. They are produced by members of the genus Caedibacter, gram-negative rod-shaped endosymbionts of paramecia and e.g. the free-living bacteria Hydrogenophaga taeniospiralis and Acidovorax avenae. The phylogenetic relationship even between the members of the genus Caedibacter is quite low: C. taeniospiralis belongs to the Gammaproteobacteria and is related to Francisella tularensis, C. caryophilus is affiliated with the Alphaproteobacteria and clusters with the obligate endosymbiont Holospora. In the case of C. taeniospiralis 51, the genetic determinants of R-body synthesis are located on a plasmid, whereas in other strains like 7 and 562 it looks like phage particles are involved in their production. In the present study, we investigated C. caryophilus, endosymbiont of Paramecium caudatum. Separation of C. caryophilus cells was performed by Percoll™ density gradient centrifugation. The isolated DNA was separated by pulsed-field gel electrophoresis and it was possible to visualize several bands referring to one or more extrachromosomal elements. A small gene library of these extrachromosomal elements was constructed and we already identified transposition-related genes; interestingly, similar genes were reported also on the plasmid of C. taeniospiralis 51.

We characterized nine macronuclear tubulin gene minichromosomes in the hypotrich ciliate Euplotidium itoi: two α-tubulin genes, five β-tubulin genes, and two γ-tubulin genes. In particular, one peculiar β-tubulin gene was identified. It shows only 81.5% amino acid similarity to the other characterized β-tubulin types and an UAG stop codon in frame. Based on this result we started to analyse stop codon usage in Euplotidium itoi macronuclear tubulin genes and some unrelated macronuclear minichromosomes. Most of the ciliates independently evolved diverse non-canonical genetic codes. Differences to the standard code consist in the reassignment of one or two of the three standard stop codons to encode a particular amino acid. Class Spirotrichea represents a particular case comprising two groups of organisms that do not share any stop codon. In oxytrichids, UAR is translated as glutamine and UGA is the single stop codon. This is in contrast to the genus Euplotes, close relatives of Euplotidium, in which UAR encodes a stop signal and UGA is translated as cysteine. Fourteen complete genes were characterized in Euplotidium itoi; in all cases UAA revealed as the single triplet used to code a stop, UGA was never and UAG rarely observed in frame. The UAG stop codon was observed in the peculiar type 3 β-tubulin gene, in two dynein partial sequences and in some other putative genes. We could confirm the active expression of three of these genes, the unusual β-tubulin, a putative lipase and a possible steroid-binding protein, by RT-PCR. Finally, a detailed analysis on the structure of the macronuclear tubulin genes, including nucleotide and amino acid differences among tubulin gene paralogs, polyadenylation sites, putative chromosome fragmentation sites, ATGC-content in coding and non-coding regions, telomere sequence and length will be presented.

Verrucomicrobiales are a newly proposed, deep branched division of bacteria from which only a handful of cultured isolates are available. Culture-independent analysis of environmental samples shows that members of Verrucomicrobiales are widespread and can be divided into several subdivisions. Symbiotic species of Euplotidium (Ciliophora) and Nematodes, epixenosomes and Xiphinemobacter spp. respectively, are according to the 16S sequences also members of this group. Up to now, only minimal sequence data mainly focused on 16S rRNA gene sequences are available for this group of organisms. The phylogenetic relatives of Verrucomicrobiales are still unclear with Chlamydiales and Planctomycetes being the most probable candidates. The present study describes preliminary results on phylogenetic relationships of Verrucomicrobiales by using protein-coding genes like Hsp70 and RpoC/B. The species which are used for this study are Prosthecobacter vanneervenii, P. debontii, Opitutus sp.VeGlc2, O. terrae and epixenosomes. The genes were amplified with designed consensus-degenerated primers and results are available on the Hsp70. The sequences show several differences within the group. Verrucomicrobium and Prosthecobacter sequences cluster together, but in P. debontii, two sequences were found, and the second one appears quite divergent. Also the two Opitutus genes are quite atypical and cluster together in a deep position. Preliminary attempt was performed on Euplotidium-epixenosome complex. However, amplified and cloned sequences revealed to be closely related to ciliate sequences. Further attempt will be performed applying microtiter-plate-subtractive-hybridization to characterize epixenosome genes.

Tubulins are typical eucaryotic genes, which have recently been found in the free-living bacteria Prosthecobacter (Verrucomicrobiales). Nevertheless, microtubule-like structures were never observed in Prostecobacter, whereas they were reported in epixenosomes which are symbiotic Verrucomicrobiales of the ciliate Euplotidium. In the present work, an extensive search for tubulin like genes was performed with several combinations of PCR-primers, designed according to different criteria. Although some combinations of these primers are able to amplify simultaneously eukaryotic α- and β-tubulin as well as Prosthecobacter tubulin genes, no bacterial-like tubulin genes were identified in other Verrucomicrobiales species. Because of host derived contaminations, even a subtractive PCR approach, using microtiter-plate-subtractive-hybridization, was used with the epixenosomes. The characterization of the genomic environment of the Prosthecobacter tubulin genes was performed by gene walking. The sequences obtained from P. debontii revealed a so-far undiscovered gene duplication. The bacterial tubulin operon looks conserved among Prosthecobacter species and includes an α-tubulin, a β-tubulin and a kinesin like protein. Upstream of two operons a repetetive sequence could be found which could be a hint for horizontal gene transfer. The analysis of the genomic environments of the tubulin genes provided open reading frames, which have homology to Verrucomicrobium spinosum genes (ongoing genome project data), whereas up to now the blast search did not reveal tubulin-like genes in this organism. Present results suggest, that the Prosthecobacter tubulin genes are possibly of recent origin, maybe acquired by horizontal gene transfer, or lost by other so far studied Verrucomicrobiales. The origin and nature of microtubular genetic determinants from epixenosomes remains open.

The genus Listeria contains the two pathogenic species Listeria monocytogenes and Listeria ivanovii and the four apparently apathogenic species Listeria innocua, Listeria seeligeri, Listeria welshimeri, and Listeria grayi. Pathogenicity of the former two species is enabled by an approximately 9 kb virulence gene cluster which is also present in a modified form in L. seeligeri. For all Listeria species, the sequence of the virulence gene cluster locus and its flanking regions was either determined in this study or assembled from public databases. Furthermore, some virulence-associated internalin loci were compared among the six species. Phylogenetic analyses were performed on a data set containing the sequences of prs, ldh, vclA, and vclB (all directly flanking the virulence gene cluster), as well as the iap gene and the 16S and 23S-rRNA coding genes which are located at different sites in the listerial chromosomes. L. grayi represents the deepest branch within the genus. The remaining five species form two groupings which have a high bootstrap support and which are consistently found by using different treeing methods. One lineage represents L. monocytogenes and L. innocua, while the other contains L. welshimeri, L. ivanovii and L. seeligeri, with L. welshimeri forming the deepest branch. Based on this perception, we tried to reconstruct the evolution of the virulence gene cluster. Since no traces of lateral gene transfer events could be detected the most parsimonious scenario is that the virulence gene cluster was present in the common ancestor of L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri and L. welshimeri and that the pathogenic capability has been lost in two separate events represented by L. innocua and L. welshimeri. This hypothesis is also supported by the location of the putative deletion breakpoints of the virulence gene cluster within L. innocua and L. welshimeri.

The ARB (from Latin arbor, tree) project was initiated almost 10 years ago. The ARB program package comprises a variety of directly interacting software tools for sequence database maintenance and analysis which are controlled by a common graphical user interface. Although it was initially designed for ribosomal RNA data, it can be used for any nucleic and amino acid sequence data as well. A central database contains processed (aligned) primary structure data. Any additional descriptive data can be stored in database fields assigned to the individual sequences or linked via local or worldwide networks. A phylogenetic tree visualized in the main window can be used for data access and visualization. The package comprises additional tools for data import and export, sequence alignment, primary and secondary structure editing, profile and filter calculation, phylogenetic analyses, specific hybridization probe design and evaluation and other components for data analysis. Currently, the package is used by numerous working groups worldwide.

Based on comparative analysis of 16S rRNA gene sequences, two oligonucleotide probes for in situ detection of all members of the genus Listeria were designed. These probes allowed fast and reliable in situ detection of Listeria spp. even in complex samples like raw milk. Almost full-length iap (invasion-associated protein) gene sequences were determined for 69 Listeria monocytogenes strains of all 13 known serotypes. A comparison of these sequences revealed that the L. monocytogenes strains can be grouped into three distinct genotypes. These clusters correlate well with distinct serotypes. Thus, strains of serotypes b and d belong to genotype I, a and c to genotype II, and 4a and 4c, which are rarely isolated from humans, group together within genotype III. These results could be corroborated by further comparative sequence analysis of genes encoding two phospholipases - plcA and plcB. Based on the iap gene sequences, a highly specific and reproducible competitive PCR detection method was developed. Primer pairs targeting genotype-specific regions of the iap gene were designed. The amplification of non-specific PCR products from DNA of non-target strains was prevented by adding competitive primers. By applying this method, the rapid and reliable distinction of the three L. monocytogenes genotypes was possible.

For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema - and Desulfomonile -like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase ( dsrAB ).

A GFPmut3b-tagged derivative of broad host-range plasmid RP4 was used to monitor the conjugative transfer of the plasmid from a Pseudomonas putida donor strain to indigenous bacteria in activated sludge. Transfer frequencies were determined to be in the range of 4 x 10(-6) to 1 x 10(-5) transconjugants per recipient. In situ hybridisation with fluorescently labeled, rRNA-targeted oligonucleotides was used to phylogenetically affiliate the bacteria that had received the plasmid.

A modified genomic DNA subtraction hybridization approach for the design of strain specific PCR systems was developed and evaluated. The method is based on the magnetic separation of homologous and heterologous hybrids of amplified reference (subtracter) and target (probe) DNA fragments from probe specific fragments. The sequence data of the latter fragments were used for diagnostic primer design. Strain diagnostic PCR systems were designed and evaluated for Lactococcus lactis subsp. lactis strains WS1678, NIZO 3F27, WS1684 and NIZO L5K5I. At least one of the primers may be used as a strain specific hybridization probe.

Tetrahymena sp. was found to graze extensively on Serratia liquefaciens MG1 swim cells (1.5-3 microns long rods) resulting in the rapid elimination of the bacterial strain. However, when S. liquefaciens cells are exposed to certain surfaces they differentiate into elongated, highly motile swarm cells and these cells were found to be grazing-resistant provided their length exceeded 15 microns.

Comparative sequence analysis of small subunit rRNA is currently one of the most important methods for the elucidation of bacterial phylogeny as well as bacterial identification. Phylogenetic investigations targeting alternative phylogenetic markers such as large subunit rRNA, elongation factors, and ATPases have shown that 16S rRNA-based trees reflect the history of the corresponding organisms globally. However, in comparison with three to four billion years of evolution the phylogenetic information content of these markers is limited. Consequently, the limited resolution power of the marker molecules allows only a spot check of the evolutionary history of microorganisms. This is often indicated by locally different topologies of trees based on different markers, data sets or the application of different treeing approaches. Sequence peculiarities as well as methods and parameters for data analysis were studied with respect to their effects on the results of phylogenetic investigations. It is shown that only careful data analysis starting with a proper alignment, followed by the analysis of positional variability, rates and character of change, testing various data selections, applying alternative treeing methods and, finally, performing confidence tests, allows reasonable utilization of the limited phylogenetic information.

Summary A highly efficient, rapid and reliable PCR-based method for distinguishing the subspecies and biovar of Lactococcus lactis (Lactococcus lactis subsp. lactis, L. lactis subsp. lactis biovar diacetylactis and L. lactis subsp. cremoris) is described. Primers complementary to positions in the histidine biosynthesis operon with distinct specificities have been constructed. PCR analysis with extracted DNA or with cells of different Lactococcus lactis strains resulted in specific fragments which could be visualized after agarose gel electrophoresis. The length polymorphisms of the PCR fragments allowed a clear distinction of the L. lactis subspecies and biovar. In our study we have also included strains which in contrast to their 16S rRNA sequence belong to a different subspecies as determined by their phenotype. The results of the PCR assay agree perfectly well with the identification based on the 16S rRNA analysis.

Summary Chemotaxonomic studies were performed on strains of lactobacilli of uncertain taxonomic position isolated from spoiled beer. Five strains were found to be distinct from other Lactobacillus species. Their morphology and physiology agree with that of a Lactobacillus species which was previously designated “Lactobacillus lindneri” . The separate species status of these strains was confirmed by comparative 16S rRNA gene sequence analysis. We propose L. lindneri as a valid species of the genus Lactobacillus . Strain DSM 20690 is proposed as the type strain. An rRNA targeted oligonucleotide probe allows a reliable identification of L. lindneri .

The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail.

Summary 23S rRNA genes of 17 strains representing the α, s, γ, δ and µ subclasses of the Proteobacteria were completely sequenced. The sequences were aligned to about 120 published as well as unpublished complete or almost complete primary structures of 23S rRNAs from other members of the domain Bacteria representing all known phyla. Primary and higher order structure analyses revealed remarkable differences of predicted 23S rRNA structures from members of the different subclasses. A phylogenetic tree reflecting the relationships among proteobacteria was reconstructed based on 23S rRNA sequence comparison. The topology of the tree is similar to that of a tree based on an equivalent 16S rRNA sequence data set.

Molecular phylogeny increasingly supports the understanding of organismal relationships and provides the basis for the classification of microorganisms according to their natural affiliations. Comparative sequence analysis of ribosomal RNAs or the corresponding genes currently is the most widely used approach for the reconstruction of microbial phylogeny. The highly and less conserved primary and higher order structure elements of rRNAs document the history of microbial evolution and are informative for definite phylogenetic levels. An optimal alignment of the primary structures and a careful data selection are prerequisites for reliable phylogenetic conclusions. rRNA based phylogenetic trees can be reconstructed and the significance of their topologies evaluated by applying distance, maximum parsimony and maximum likelihood methods of phylogeny inference in comparison, and by fortuitous or directed resampling of the data set. Phylogenetic trees based on almost equivalent data sets of bacterial 23S and 16S rRNAs are in good agreement and their overall topologies are supported by alternative phylogenetic markers such as elongation factors and ATPase subunits. Besides their phylogenetic information content, the differently conserved primary structure regions of rRNAs provide target sites for specific hybridization probes which have been proven to be powerful tools for the identification of microbes on the basis of their phylogenetic relationships.

Comparative sequence analyses were performed on 14 genes encoding bacterial elongation factors EF-Tu and 7 genes encoding the beta-subunit of bacterial F1F0 type ATP-synthases. The corresponding predicted amino acid sequences were compared with published primary structures of homologous molecules. Phylogenetic trees were reconstructed from both data sets of aligned protein sequences and from an equivalent selection of 16S rRNA sequences by applying distance matrix and maximum parsimony methods. The EF-Tu data were in very good agreement with the rRNA data, although the resolution within the EF-Tu tree was reduced at certain phylogenetic levels. The resolution power of the ATPase beta-subunit sequence data were more reduced than those of the EF-Tu data. In comparison with the 16S rRNA tree there are minor differences in the order of adjacent branchings within the ATPase beta-subunit tree.

The phylogenetic position of Caedibacter caryophila, a so far noncultured killer symbiont of Paramecium caudatum, was elucidated by comparative sequence analysis of in vitro amplified 16S rRNA genes (rDNA). C. caryophila is a member of the alpha subclass of the Proteobacteria phylum. Within this subclass C. caryophila is moderately related to Holospora obtusa, which is another obligate endosymbiont of Paramecium caudatum, and to Rickettsia. A 16S rRNA targeted specific hybridization probe was designed and used for in situ detection of C. caryophila within its host cell. Comparison of the 16S rDNA primary structure of C. caryophila with homologous sequences from other bacteria revealed an unusual insertion of 194 base pairs within the 5'-terminal part of the corresponding gene. The intervening sequence is not present in mature 16S rRNA of C. caryophila. It was demonstrated that C. caryophila contained fragmented 16S rRNA.

Summary Ribotyping and the randomly amplified polymorphic DNA (RAPD) technique were evaluated as tools for epidemiological studies of Vibrio vulnificus . Ribopatterns of DNA digested with four restriction enzymes and hybridized with two oligonucleotides complementary to highly conserved sequences in the 16S and 23S rRNA genes showed different levels of discrimination among the strains tested. In addition, the presence of eight to ten rRNA operons was revealed in Vibrio strains. Strains of V. vulnificus biotypes 1 and 2 could be differentiated by random amplification of DNA with M13, T3 or T7 universal primers. Compared with ribotyping, RAPD appeared to be a faster method for diagnostic identification of V. vulnificus biotypes since a single colony could be used as a source of DNA template and the final result was obtained within 24 h after sampling. Further, RAPD analysis allowed the identification of individual strains.

A gene library of the hyperthermophilic bacterium Thermotoga maritima strain MSB8 was constructed in Escherichia coli. Two non-related T. maritima chromosomal DNA fragments were physically characterized. They conferred the synthesis of thermostable X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside)-hydrolysing activity upon the host organism. The biochemical properties of the recombinant enzymes indicated that genes for a β-galactosidase (BgaA) and a broad-specificity β-glucosidase (Bg1A) had been isolated. The genes were desiignted bgaA and bglA, respectively. According to analytical size exclusion chromatography data, BgaA and BglA had native molecular masses of approximately 240 kDa and 95 kDa, respectively. Both enzymes apparently have dimeric subunit structure. An additional β-glucosidase (designated BglB) activity, clearly distinct from BglA in terms of substrate specificity, could be detected in a crude extract of T. maritima.

The “All-Species Living Tree Project” (LTP) provides the scientific community with a useful taxonomic tool consisting of a curated database of type strain sequences, a universal and optimized alignment and a single phylogenetic tree harboring all the type strains of the hitherto classified species [33]. On the website http://www.arb-silva.de/projects/living-tree an update has been regularly maintained by including the 1301 new descriptions that have appeared in the validation and notification lists of the IJSEM journal. The topology of the 16S rRNA-based tree was validated with a detailed comparison against a collection of taxa-specific and broad-range trees made using different approaches, subsets of sequences and alignments. Seven percent of the classified species is still missing, as their type strains do not have a good quality SSU sequence. In addition, a new database of type strains for which adequate 23S rRNA entries existed in public repositories was built. Among the 8602 species with validly published names until February 2010, we were able to find good quality LSU representatives for 792 type strains, whereas around 91% of the complete catalogue still remains unsequenced. Despite the scarce representation of some groups in LSU databases, we have devised a highly optimized alignment and a reliable LSU tree in order to set up a stable phylogenetic starting point for taxonomic purposes. The current release corresponds to the fourth update of the project (LTPs102), and contains additional features which increase usability and compatibility. Use the contact address living-tree@arb-silva.de to provide additional input for the development of this taxonomic tool., Spanish Ministry of Science and Innovation (Project Consolider Ingenio 2010 CE-CSD2007-0005 co-financed with FEDER funding)

An insertion of about 100 bases within the central part of the 23S rRNA genes was found to be a phylogenetic marker for the bacterial line of descent of Gram-positive bacteria with a high DNA G + C content. The insertion was present in 23S rRNA genes of 64 strains representing the major phylogenetic groups of Gram-positive bacteria with a high DNA G+C content, whereas it was not found in 23S rRNA genes of 55 (eu)bacteria representing Gram-positive bacteria with a low DNA G + C content and all other known (eu)bacterial phyla. The presence of the insertion could be easily demonstrated by comparative gel electrophoretic analysis of in vitro-amplified 23S rDNA fragments, which contained the insertion. The nucleotide sequences of the amplified fragments were determined and sequence similarities of at least 44% were found. The overall similarity values are lower than those of 16S and 23S rRNA sequences of the particular organism. Northern hybridization experiments indicated the presence of the insertion within the mature 23S rRNA of Corynebacterium glutamicum.

A gene probe (nucleic acid probe) is a single-stranded nucleic acid fragment that interacts with a complementary sequence of a target nucleic acid. The test is based upon the principles of nucleic acid hybridization reactions. Different assay formats (dot-blot, colony, whole-cell hybridizations) can be applied. Gene probes can be used for the rapid and specific identification of microorganisms. The phylogenetic identification and in situ detection of uncultured bacteria will be discussed.

Recently, the features of a group of strains isolated from Dead Sea enrichments obtained in 1936 by one of us (B. E. Volcani) were described. They were gram-positive, moderately halophilic, spore-forming rods, and were placed in a new species, Bacillus marismortui. At the same time, the new genus Salibacillus was proposed for the halophilic species Bacillus salexigens. B. marismortui and Salibacillus salexigens have similar phenotypic characteristics and the same peptidoglycan type. Phylogenetic analysis based on 16S rRNA sequence comparisons showed that they are sufficiently closely related (96.6% similarity) as to warrant placement in the same genus. However, DNA-DNA hybridization experiments showed that they constitute two separate species (41% DNA similarity). Therefore the reclassification of Bacillus marismortui as Salibacillus marismortui comb. nov. is proposed.

Comparative sequence analysis of 16S ribosomal (r)RNAs or DNAs of Bacillus alvei, B. laterosporus, B. macerans, B. macquariensis, B. polymyxa and B. stearothermophilus revealed the phylogenetic diversity of the genus Bacillus. Based on the presently available data set of 16S rRNA sequences from bacilli and relatives at least four major "Bacillus clusters" can be defined: a "Bacillus subtilis cluster" including B. stearothermophilus, a "B. brevis cluster" including B. laterosporus, a "B. alvei cluster" including B. macerans, B. maquariensis and B. polymyxa and a "B. cycloheptanicus branch".

The entire Escherichia coli lactose operon was inserted into an E. coli/Corynebacterium glutamicum shuttle vector and introduced into the gram-positive host organism C. glutamicum R 163. Recombinant C. glutamicum strains carrying the lac genes downstream of an efficient promoter displayed rapid growth with lactose as the sole source of carbon. Two prerequisites were necessary to obtain good growth of C. glutamicum R 163 on lactose: presence of the lacY gene in addition to lacZ and an appropriate promoter for efficient transcription in C. glutamicum. The galactose moiety of lactose was not utilized but accumulated in the culture broth. C. glutamicum strains carrying only the lacZ (beta-galactosidase) gene but not lacY (lactose permease) were not able to grow in lactose minimal medium.

The signing authors together with the journal Systematic and Applied Microbiology (SAM) have started an ambitious project that has been conceived to provide a useful tool especially for the scientific microbial taxonomist community. The aim of what we have called >The All-Species Living Tree> is to reconstruct a single 16S rRNA tree harboring all sequenced type strains of the hitherto classified species of Archaea and Bacteria. This tree is to be regularly updated by adding the species with validly published names that appear monthly in the Validation and Notification lists of the International Journal of Systematic and Evolutionary Microbiology. For this purpose, the SAM executive editors, together with the responsible teams of the ARB, SILVA, and LPSN projects (www.arb-home.de, www.arb-silva.de, and www.bacterio.cict.fr, respectively), have prepared a 16S rRNA database containing over 6700 sequences, each of which represents a single type strain of a classified species up to 31 December 2007. The selection of sequences had to be undertaken manually due to a high error rate in the names and information fields provided for the publicly deposited entries. In addition, from among the often occurring multiple entries for a single type strain, the best-quality sequence was selected for the project. The living tree database that SAM now provides contains corrected entries and the best-quality sequences with a manually checked alignment. The tree reconstruction has been performed by using the maximum likelihood algorithm RAxML. The tree provided in the first release is a result of the calculation of a single dataset containing 9975 single entries, 6728 corresponding to type strain gene sequences, as well as 3247 additional high-fquality sequences to give robustness to the reconstruction. Trees are dynamic structures that change on the basis of the quality and availability of the data used for their calculation. Therefore, the addition of new type strain sequences in further subsequent releases may help to resolve certain branching orders that appear ambiguous in this first release. On the web sites: www.elsevier.de/syapm and www.arb-silva.de/living-tree, the All-Species Living Tree team will release a regularly updated database compatible with the ARB software environment containing the whole 16S rRNA dataset used to reconstruct >The All-Species Living Tree>. As a result, the latest reconstructed phylogeny will be provided. In addition to the ARB file, a readable multi-FASTA universal sequence editor file with the complete alignment will be provided for those not using ARB. There is also a complete set of supplementary tables and figures illustrating the selection procedure and its outcome. It is expected that the All-Species Living Tree will help to improve future classification efforts by simplifying the selection of the correct type strain sequences. For queries, information updates, remarks on the dataset or tree reconstructions shown, a contact email address has been created (living-tree@arb-silva.de). This provides an entry point for anyone from the scientific community to provide additional input for the construction and improvement of the first tree compiling all sequenced type strains of all prokaryotic species for which names had been validly published. © 2008 Elsevier GmbH. All rights reserved., The authors want to acknowledge the Max Planck Society and the Project Consolider (CE-CSD2007-0005) of the CICYT for funding support

The late 19th century was the beginning of bacterial taxonomy and bacteria were classified on the basis of phenotypic markers. The distinction of prokaryotes and eukaryotes was introduced in the 1960s. Numerical taxonomy improved phenotypic identification but provided little information on the phylogenetic relationships of prokaryotes. Later on, chemotaxonomic and genotypic methods were widely used for a more satisfactory classification. Archaea were first classified as a separate group of prokaryotes in 1977. The current classification of Bacteria and Archaea is based on an operational-based model, the so-called polyphasic approach, comprised of phenotypic, chemotaxonomic and genotypic data, as well as phylogenetic information. The provisional status Candidatus has been established for describing uncultured prokaryotic cells for which their phylogenetic relationship has been determined and their authenticity revealed by in situ probing. The ultimate goal is to achieve a theory-based classification system based on a phylogenetic/evolutionary concept. However, there are currently two contradictory opinions about the future classification of Bacteria and Archaea. A group of mostly molecular biologists posits that the yet-unclear effect of gene flow, in particular lateral gene transfer, makes the line of descent difficult, if not impossible, to describe. However, even in the face of genomic fluidity it seems that the typical geno- and phenotypic characteristics of a taxon are still maintained, and are sufficient for reliable classification and identification of Bacteria and Archaea. There are many well-defined genotypic clusters that are congruent with known species delineated by polyphasic approaches. Comparative sequence analysis of certain core genes, including rRNA genes, may be useful for the characterization of higher taxa, whereas various character genes may be suitable as phylogenetic markers for the delineation of lower taxa. Nevertheless, there may still be a few organisms which escape a reliable classification.

A 1330 base-pair fragment of a 16S rRNA gene has been amplified, cloned and sequenced. Comparison to other 16S rRNA sequences of eubacteria showed that P. niger represents a deep branch within the subdivision "Gram-positive with Gram-negative cell walls". It is not related to peptostreptococci, representatives of this genus studied so far are more closely related to clostridia.

Escherichia coli K-12, B, C and W strains are the most frequently used bacterial safety and laboratory strains. Lineage-specific DNA fragments were detected by microplate subtractive hybridization and utilized to create a fast differentiation method using a single PCR reaction to differentiate clearly the four lineages and separate them from pathogenic variants. The method has been evaluated on a comprehensive selection of widely used laboratory strains and a variety of pathogenic E. coli representatives. In addition, in silico analysis on all available E. coli genomes and the genomes of the close relatives Shigella and Salmonella confirmed the reliability of the proposed method. A fast identification and differentiation of E. coli safety strains by Multiplex-PCR is a useful tool for researchers and companies to check and monitor their reference stocks.

Prosthecobacter, one of the few cultivable representatives of the bacterial phylum Verrucomicrobia, is of increasing interest to the scientific community due to the presence of tubulin genes in its genome and the apparent absence of the bacterial homologue FtsZ that is normally involved in prokaryotic cell division. These findings suggested the possibility of a vicarious takeover of the FtsZ function through these novel tubulins and opened new scenarios on the possible evolution of bacterial cytoskeleton and cell division. In the present manuscript, we report the characterization of ftsZ and ftsA homologues in different Prosthecobacter species that also possess tubulin genes. Based on these findings, we propose an FtsZ-based cell division mechanism in Verrucomicrobia. The analysis of available genome data of Verrucomicrobia suggests that tubulins are not a feature common to all members of this phylum. Therefore, it can be assumed that Prosthecobacter acquired tubulins through horizontal gene transfer. The functional role of tubulins in Prosthecobacter remains enigmatic.

Eu.bac.te.ri.a'ce.ae. N.L. neut. n. Eubacterium type genus of the family; suff. -aceae ending denoting family; N.L. fem. pl. n. Eubacteriaceae the Eubacterium family. Firmicutes / “Clostridia” / Clostridiales / “Eubacteriaceae” Keywords: Eubacteriaceae fam. nov; Eubacterium; Clostridia class. nov.; Clostridiales

For detection of most members of the Enterococcaceae, the specificity of a novel oligonucleotide microarray (ECC-PhyloChip) consisting of 41 hierarchically nested 16S or 23S rRNA gene-targeted probes was evaluated with 23 pure cultures (including 19 Enterococcus species). Target nucleic acids were prepared by PCR amplification of a 4.5-kb DNA fragment containing large parts of the 16S and 23S rRNA genes and were subsequently labeled fluorescently by random priming. Each tested member of the Enterococcaceae was correctly identified on the basis of its unique microarray hybridization pattern. The evaluated ECC-PhyloChip was successfully applied for identification of Enterococcus faecium and Enterococcus faecalis in artificially contaminated milk samples demonstrating the utility of the ECC-PhyloChip for parallel identification and differentiation of Enterococcus species in food samples.

Microbiology has entered the molecular age. Almost every month another complete bacterial genome sequence is published (e.g., Fleischmann et al., 1995; Cole et al., 1998), and it is now routine to start the classification of a newly isolated microorganism with the determination and comparative analysis of at least one nucleic acid sequence. Clearly, the most commonly used molecule for this purpose is the ribonucleic acid of the small subunit of the ribosome, the 16S rRNA of Bacteria and Archaea. The high information content of nucleic acid sequences can, in principle, be accessed by two techniques. One is sequencing followed by comparative sequence analysis; the second is hybridization with nucleic acid probes.

Summary Almost complete 16S rRNA gene primary structures of Pelobacter acetylenicus strain WoAcyl (DSM 2348) and Pelobacter propionicus strain OttBd14 (DSM2379) were determined by direct sequencing of in vitro amplified DNA. Both species are members of the δ-subclass of Proteobacteria. Pelobacter acetylenicus is phylogenetically closer related to Pelobacter acidigallici, Pelobacter carbinolicus, Pelobacter venetianus and to Desulfuromonas species than to Pelobacter propionicus.

A critical evaluation of published and own taxonomic and phylogenetic studies on Vibrio pelagius showed substantial diversity of strains received as type strains from various Culture Collections. The comparison of data based upon 16S rRNA sequence analyses, earlier genomic DNA-DNA similarity studies as well as physiological investigations and the original description indicate that Vibrio pelagius strains CECT 4202T and ATCC 25916T really represent the originally described type species whereas strains NCIMB 1900T and CIP 102762T highly likely are representatives of Vibrio natriegens.

The recently proposed reorganization of the order Chlamydiales and description of new taxa are broadening our perception of this once narrowly defined taxon. We have recovered four strains of gram-negative cocci endosymbiotic in Acanthamoeba spp., representing 5% of the Acanthamoeba sp. isolates examined, which displayed developmental life cycles typical of members of the Chlamydiales . One of these endosymbiont strains was found stably infecting an amoebic isolate recovered from a case of amoebic keratitis in North America, with three others found in acanthamoebae recovered from environmental sources in North America (two isolates) and Europe (one isolate). Analyses of nearly full-length 16S rRNA gene sequences of these isolates by neighbor joining, parsimony, and distance matrix methods revealed their clustering with other members of the Chlamydiales but in a lineage separate from those of the genera Chlamydia , Chlamydophila , Simkania , and Waddlia (sequence similarities, Parachlamydia acanthamoebae (sequence similarities, 91.2 to 93.1%). With sequence similarities to each other of 91.4 to 99.4%, these four isolates of intra-amoebal endosymbionts may represent three distinct species and, perhaps, new genera within the recently proposed family Parachlamydiaceae . Fluorescently labeled oligonucleotide probes targeted to 16S rRNA signature regions were able to readily differentiate two groups of intra-amoebal endosymbionts which corresponded to two phylogenetic lineages. These results reveal significant phylogenetic diversity occurring among the Chlamydiales in nontraditional host species and supports the existence of a large environmental reservoir of related species. Considering that all described species of Chlamydiales are known to be pathogenic, further investigation of intra-amoebal parachlamydiae as disease-producing agents is warranted.

Summary DNA-DNA hybridization studies under stringent conditions and physiological data provide sufficient evidence for conferring species rank on Streptococcus salivarius subsp. thermophilus.

Acanthamoebae are ubiquitous soil and water bactivores which may serve as amplification vehicles for a variety of pathogenic facultative bacteria and as hosts to other, presently uncultured bacterial endosymbionts. The spectrum of uncultured endosymbionts includes gram-negative rods and gram-variable cocci, the latter recently shown to be members of the Chlamydiales . We report here the isolation from corneal scrapings of two Acanthamoeba strains that harbor gram-negative rod endosymbionts that could not be cultured by standard techniques. These bacteria were phylogenetically characterized following amplification and sequencing of the near-full-length 16S rRNA gene. We used two fluorescently labelled oligonucleotide probes targeting signature regions within the retrieved sequences to detect these organisms in situ. Phylogenetic analyses demonstrated that they displayed 99.6% sequence similarity and formed an independent and well-separated lineage within the Rickettsiales branch of the alpha subdivision of the Proteobacteria . Nearest relatives included members of the genus Rickettsia , with sequence similarities of approximately 85 to 86%, suggesting that these symbionts are representatives of a new genus and, perhaps, family. Distance matrix, parsimony, and maximum-likelihood tree-generating methods all consistently supported deep branching of the 16S rDNA sequences within the Rickettsiales . The oligonucleotide probes displayed at least three mismatches to all other available 16S rDNA sequences, and they both readily permitted the unambiguous detection of rod-shaped bacteria within intact acanthamoebae by confocal laser-scanning microscopy. Considering the long-standing relationship of most Rickettsiales with arthropods, the finding of a related lineage of endosymbionts in protozoan hosts was unexpected and may have implications for the preadaptation and/or recruitment of rickettsia-like bacteria to metazoan hosts.

Natural enrichments of magnetic bacteria from the Itaipu lagoon near Rio de Janeiro were dominated by coccoid-to-ovoid morphotypes that produced unusually large magnetosomes. To determine the phylogenetic position of these unusual microorganisms, 16S rRNA genes were retrieved from bacteria magnetically separated from sediment of the Itaipu lagoon by in vitro amplification and cloning of PCR products into a plasmid vector. Partial sequencing of the obtained clones revealed two clusters of closely related sequences affiliated to a distinct lineage consisting exclusively of magnetic bacteria within the alpha-subclass of Proteobacteria. For a detailed phylogenetic analysis, several almost complete sequences of the 16S rRNA genes were determined. One representative clone of each cluster provided a PCR template for the in vitro transcription of group-specific polynucleotide probes complementary to a variable region of the 16S rRNA molecule. At least three different morphotypes of magnetic bacteria were reliably identified by post-embedding hybridization of ultra-thin sections. Electron microscopic analyses of hybridized cells enabled for the first time a detailed description of the morphological variety and ultrastructure of phylogenetically identified, uncultured magnetic bacteria. Two distinct coccoid bacteria were identified by the transcript probe complementary to the 16S rRNA sequence mabrj12, whereas the probe complementary to the sequence mabrj58 allowed the identification of an ovoid morphotype that displayed magnetosomes with the largest volumes observed to date.

Simultaneous in situ analysis of the structure and function of bacterial cells present within complex communities is a key for improving our understanding of microbial ecology. A protocol for the in situ identification of Listeria spp. using fluorescently tagged, rRNA-targeted oligonucleotide probes was developed. Ethanol fixation and enzymatic pretreatment with lysozyme and proteinase K were used to optimize whole cell hybridization of exponential phase and stationary phase Listeria spp. cells. In parallel, transcript probes carrying multiple digoxigenin molecules were combined with anti-digoxigenin Fab antibody fragments labeled with horseradish peroxidase to detect, via the catalytic deposition of fluorescein-tyramide, the iap-mRNA in single Listeria monocytogenes cells. The iap gene encodes the associated virulence factor p60. Application of the new signal amplification technique resulted in strong signals comparable in intensity to those obtained with fluorescently labeled rRNA-targeted oligonucleotide probes.

Ribosomal ribonucleic acids are excellent marker molecules for the elucidation of bacterial phylogeny; they also provide useful target sites for identification and detection with nucleic acid probes. Based on the currently available 16S rRNA sequence data, bacteria of the rhizobial phenotype (plant nodulation, nitrogen fixation) are members of three moderately related phylogenetic sub-groups of the α-subclass of the Proteobacteria: i.e. the rhizobia group, the bradyrhizobia group, and the azorhizobia group. All rhizobia, azo-, brady-, meso-and sinorhizobia are closely related to and in some cases phylogenetically intermixed with, non-symbiotic and/or non-nitrogen-fixing bacteria. Especially in the case of Bradyrhizobium japonicum strains, the 16S rRNA sequence data indicate substantial heterogeneity. Specific probe design and evaluation are discussed. A multiprobe concept for resolving specificity problems with group specific probes is presented. In situ identification with group specific probes of rhizobia in cultures as well as rhizobia and cyanobacteria within plant material is shown.

16S rRNA gene libraries were prepared by polymerase chain reaction amplification and cloning from soil samples taken periodically from a field with genetically modified plants. Sequence analyses of the cloned rDNAs indicated that 140 of them clustered apart from known bacterial phyla. Based on 31 full sequences a new phylum could be defined. It includes Holophaga foetida, 'Geothrix fermentans' and Acidobacterium capsulatum as the only cultured species so far. Therefore, this line of descent was named the Holophagal Acidobacterium phylum. About 50 published partial sequences of cloned rDNAs retrieved from soil, freshwater sediments or activated sludge from different continents indicate the occurrence of further representatives of this phylum. Two specific hybridization probes were constructed for members of one of four subclusters. A careful data analysis revealed the importance and problems of identifying and dealing with artefacts such as chimeric structure when defining new phylogenetic groups based mainly upon cloned amplified rDNAs. For the first time, the presence of bacterial cells representing this group could be shown in soil, sediment, activated sludge and lake snow by in situ hybridization.

The bacterial community structure of activated sludge of a large municipal wastewater treatment plant was investigated by use of the rRNA approach. Almost-full-length genes coding for the small-subunit rRNA (rDNA) were amplified by PCR and subsequently cloned into the pGEM-T vector. Clones were screened by dot blot hybridization with group-specific oligonucleotide probes. The phylogenetic affiliations of clones were compared with the results obtained with the original sample by in situ hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes and found to be in general agreement. Twenty-five 16S rDNA clones were fully sequenced, 11 were almost fully (> 80%) sequenced, and 27 were partially sequenced. By comparative sequence analyses, the majority of the examined clones (35 of 67) could be affiliated with the beta subclass of the class Proteobacteria. The gamma and alpha subclasses of Proteobacteria were represented by 13 and 4 clones, respectively. Eight clones grouped with the epsilon group of Proteobacteria, and five clones grouped with gram-positive bacteria with a low DNA G+C content. The 16S rDNA of two clones showed similarity with 16S rDNA genes of members of the phyla Chlamydiae and Planctomyces. 16S rRNA-targeted oligonucleotide probes were designed and used for the enumeration of the respective bacteria. Interestingly, potentially pathogenic representatives of the genus Arcobacter were present in significant numbers (4%) in the activated sludge sample examined. Pairs of probes targeted to the 5' and 3' regions were used for detection of chimeric sequences by in situ hybridization. Two clones could be identified as chimera by applying such a pair of probes.

A new, phylogenetically distinct, dissimilatory, Fe(III)-reducing bacterium was isolated from surface sediment of a hydrocarbon-contaminated ditch. The isolate, designated strain PAL-1, was an obligately anaerobic, non-fermentative, motile, gram-negative vibrio. PAL-1 grew in a defined medium with acetate as electron donor and ferric pyrophosphate, ferric oxyhydroxide, ferric citrate, Co(III)-EDTA, or elemental sulfur as sole electron acceptor. PAL-1 also used proline, hydrogen, lactate, propionate, succinate, fumarate, pyruvate, or yeast extract as electron donors for Fe(III) reduction. It is the first bacterium known to couple the oxidation of an amino acid to Fe(III) reduction. PAl-1 did not reduce oxygen, Mn(IV), U(VI), Cr(VI), nitrate, sulfate, sulfite, or thiosulfate with acetate as the electron donor. Cell suspensions of PAL-1 exhibited dithionite-reduced minus air-oxidized difference spectra that were characteristic of c-type cytochromes. Analysis of the 16S rRNA gene sequence of PAL-1 showed that the strain is not related to any of the described metal-reducing bacteria in the Proteobacteria and, together with Flexistipes sinusarabici, forms a separate line of descent within the Bacteria. Phenotypically and phylogenetically, strain PAl-1 differs from all other described bacteria, and represents the type strain of a new genus and species, Geovibrio ferrireducens.