Bacterial phylogeny based on 16S and 23S rRNA sequence analysis.

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.

Quantitative determination of human IgG antibodies to the peptide subunit determinant of peptidoglycan by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative measurement of IgG antibodies to the immunodominant R-D-Ala-D-Ala-OH determinant of peptidoglycan. Synthetic peptides R-D-Ala-D-Ala-OH, revealing structural analogy with the C-terminal sequence of the antigenic determinant H-L-Ala-D-Glu(L-Lys-D-Ala-D-Ala-OH)-NH2 of peptidoglycan, were coupled covalently to albumin via their amino groups. The resulting peptidyl proteins were employed as an antigen in an ELISA for the specific detection of human IgG antibodies against the C-terminal R-D-Ala-D-Ala-OH moiety of H-L-Ala-D-Glu(L-Lys-D-Ala-D-Ala-OH)-NH2. Antigenic specificity was proved by comparing the high binding to albumin-(D-Ala-D-Ala-D-Ala-OH)9 with a lack of binding to albumin-(L-Ala-L-Ala-L-Ala-OH)13 and by appropriate inhibition studies of the ELISA. IgG, totally free from IgA and IgM, was isolated from reference serum 004, and the particular specificity was entirely found in this fraction. Quantification of the ELISA was effected by affinity chromatography. Isolated IgG was applied to an affinity column of Sepharose-[albumin-(D-Ala-D-Ala-D-Ala-OH)9]n, unbound IgG was eluted with phosphate-buffered saline and specific IgG against the C-terminal R-D-Ala-D-Ala-OH moiety of H-L-Ala-D-Glu(L-Lys-D-Ala-D-Ala-OH)-NH2 was eluted with 6 M guanidinium chloride.

Cloning and expression of the alpha-amylase gene from Bacillus stearothermophilus in several staphylococcal species.

The plasmid-coded alpha-amylase gene of Bacillus stearothermophilus (amy) was cloned in Staphylococcus carnosus using plasmid pCA43 as a vector. The amy gene was located on a 5.4-kb HindIII DNA fragment of the hybrid plasmid pamy7. When transformed into other staphylococcal species, plasmid pamy7 exhibited marked differences in the production of alpha-amylase (alpha Amy). Most active for heterospecific alpha Amy production was Staphylococcus aureus. In its culture supernatant nearly half as much alpha Amy activity was found as for the donor strain B. stearothermophilus. All staphylococcal species were able to secrete alpha Amy, since more than 80% of the enzyme activity was found in the culture supernatant. The extracellular alpha Amy of S. aureus [pamy7] was purified to homogeneity. The enzyme exhibited an Mr of approx. 58 000, an optimum activity at pH 5.3-6.3 and at 65 degrees C. Although the enzyme was stable at 65 degrees C for at least 3 h, its thermostability was not unusual. The enzymatic properties of the alpha Amy from S. aureus were similar to those previously reported for various B. stearothermophilus strains.

Reverse dot blot hybridization: a useful method for the direct identification of lactic acid bacteria in fermented food.

A rapid method for a reliable and simultaneous identification of different lactic acid bacteria in fermented food has been developed. Various 16S and 23S rRNA-targeted, species-specific oligonucleotides were applied as capture probes in a non-radioactive reverse dot blot hybridization. A simple and fast DNA extraction method in combination with in vitro amplification of rRNA gene fragments enables the direct detection of typical starter organisms without any preceding enrichment or cultivation steps. Various lactic acid bacteria occurring in cheese, yogurt, sausages, sauerkraut and sourdough could be identified at the species level within 1 day.

Differentiation of lactococci by rRNA gene restriction analysis.

Strains of the subspecies of Lactococcus lactis could be differentiated by rRNA gene restriction fragment length polymorphisms (RFLP). 16S rRNA-specific oligonucleotide as well as polynucleotide DNA probes were used for the detection of restriction fragments. In addition, a site-specific probe was designed for the intergenic spacer region of 23S and 5S rRNA genes. For all lactococcal strains the putative presence of six rRNA operons was confirmed. A non-radioactive hybridization assay was used based on hybrid detection by chemiluminescence. Specific patterns were found for any of the strains investigated. Subspecies-specific restriction fragments could be identified in addition to the strain-specific patterns.

Polynucleobacter necessarius, an obligate bacterial endosymbiont of the hypotrichous ciliate Euplotes aediculatus, is a member of the beta-subclass of Proteobacteria.

An almost full length 16S rRNA gene of the obligate bacterial endosymbiont Polynucleobacter necessarius was amplified using the polymerase chain reaction in combination with site-specific primers. The amplified DNA was directly sequenced and compared with other bacterial 16S rRNA sequences. P. necessarius belongs to the beta-subclass of Proteobacteria and shows the closest relationship to Alcaligenes eutrophus, Burkholderia solanacearum, and B. pickettii. In Proteobacteria and shows the closest relationship to Alcaligenes eutrophus, Burkholderia solanacearum, and B. pickettii. In situ hybridization with a specific oligonucleotide probe corroborated the assignment of the retrieved sequence to P. necessarius.

The phylogenetic position of Peptococcus niger based on 16S rRNA sequence studies.

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.

High efficiency electroporation of intact Corynebacterium glutamicum cells.

High-frequency electroporation of whole Corynebacterium glutamicum cells without enzymatic pretreatment was achieved. Under optimized conditions concerning growth stage, washing of cells, cell concentration and pulse parameter transformation efficiencies of far more than 10(7) transformants per microgram pWST4B plasmid DNA were reached. Using electroporation, linearised and subsequently religated plasmid as well as chimeric ligase reaction products were directly introduced into C. glutamicum with reasonable efficiencies. Electrotransformation efficiency was reduced about 10(5)-fold for plasmid DNA cycled through E. coli JM83. Restriction deficient mutants of C. glutamicum were isolated which could be efficiently transformed with foreign DNA.

The phylogenetic status of Sarcobium lyticum, an obligate intracellular bacterial parasite of small amoebae.

A 16S rRNA gene of the obligate intracellular bacterial parasite Sarcobium lyticum was amplified using the polymerase chain reaction in combination with site-specific primers. The amplified DNA was cloned, sequenced and compared with other bacterial 16S rRNA sequences. The analysis revealed that S. lyticum belongs to the gamma subclass of the Proteobacteria and shows the closest relationship to an intracellular Legionella species recovered by amoebal enrichment from the sputum of a patient with pneumonia. S. lyticum could be detected in situ with a fluorescent oligonucleotide probe by whole cell hybridization.

In situ detection of a virulence factor mRNA and 16S rRNA in Listeria monocytogenes.

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.

Serratia liquefaciens swarm cells exhibit enhanced resistance to predation by Tetrahymena sp.

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.

Monitoring the conjugal transfer of plasmid RP4 in activated sludge and in situ identification of the transconjugants.

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.

Propionigenium modestum: a separate line of descent within the eubacteria.

The complete nucleotide sequence of 16S rRNA from Propionigenium modestum was determined and compared with 380 16S rRNA sequences from representatives of all eu- and archaebacterial phyla known so far. The phylogenetic analysis of this data set indicated P. modestum to represent a new separated line of descent within the radiation of eubacterial phyla moderately related to cyanobacteria and Gram-positive bacteria with low DNA GC content.

Detection and in situ identification of representatives of a widely distributed new bacterial phylum.

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.

Relatedness and classification of Streptococcus mutans and "mutans-like" streptococci.

The "mutans-like" streptococci can be separated into five species (Streptococcus mutans, S. rattus, S. sobrinus, S. cricetus, and S. ferus) that belong to the same rRNA homology cluster. New valuable chemical characters for differentiation of the five species--such as peptidoglycan type, presence of cell wall teichoic acid, and cell wall sugar composition--are described. The peptidoglycan type and the cell wall sugar composition can be determined by rapid procedures.

How quantitative is quantitative PCR with respect to cell counts?

Quantitative diagnostic PCR systems based upon rDNA targeted primer and probe combinations were developed for the detection of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, enterococci, Staphylococcus aureus, and Staphylococcus epidermidis. Primers and probes were designed in silico using the ARB software package (TU Munich) in combination with Primer Design software of PE Applied Biosystems. Purified genomic DNA or bacterial cells of target and reference organisms were used for the evaluation of the PCR assays applying the TaqMan technique on an ABI PRISM TM 7700 Sequence Detection System (PE Applied Biosystems). Sensitive, reliable and reproducible quantification of target rDNA could be achieved applying primer-probe combinations that mediate in vitro amplification of DNA fragments smaller than 100 base pairs. Large amounts of non target DNA (1 mg per sample) remarkably affected the quantification potential of the approach resulting in an underestimation of the amounts of target DNA. One of the principal goals was to use quantitative PCR to study the correlation of gene and cell numbers depending on the growth behavior of target organisms and to explore the potential to estimate cell numbers from target DNA quantification. A clear correlation of rDNA quantification and bacterial growth was observed, however, cell numbers cannot directly be estimated from quantitative PCR data, given that the cellular genome content varies with the growth phase of the organisms. In the case of Escherichia coli the cell numbers which could be assigned to a certain number of rDNA targets varied reasonably depending upon the growth phase of batch cultures.

DNA polynucleotide probes generated from representatives of the genus Acinetobacter and their application in fluorescence in situ hybridization of environmental samples.

The application of rRNA directed polynucleotide probes carrying multiple labels facilitates the detection of target cells by fluorescence in situ hybridizations and allows specific enrichment by cell fishing. So far, exclusively RNA transcript probes have been used. To reduce the effort in the preparation of the polynucleotides and to enhance their stability, DNA probes matching a part of the highly variable domain III on the 23S rRNA were constructed by amplification of the target region using PCR. Fluorescent labeling was achieved by incorporation of Cy3-labeled desoxyribonucleotides in the amplification. DNA polynucleotide probes were constructed for the seven validly described Acinetobacter species. Amplified domain III rDNA of A. baumannii and A. calcoaceticus could be readily applied as species specific probe. In addition, rDNA fragments could be used to recognize two groups of species, one comprising A. haemolyticus, A. junii and A. radioresistens and the other one A. lwoffii and A. johnsonii. Acinetobacter baumannii cells, some of them occurring in filaments, could be detected by in situ hybridization in native samples of activated sludge.

Studies on the spectrophotometric determination of DNA hybridization from renaturation rates.

The optical method of De Ley et al. (1970) for determining DNA/DNA homologies was reexamined. Several parameters such as incubation temperature, incubation time, DNA concentration and DNA fragment length were checked and the optimal conditions established. Hybridization data of several anaerobic Gram-positive cocci were compared with values obtained by the membrane filter technique. The agreement is excellent above a degree of binding of 25-30%.

Phylogenetic and Biochemical Studies on Stomatococcus mucilaginosus.

The degree of relatedness of nine strains of Stomatococcus mucilaginosus (formerly classified as 'Micrococcus mucilaginosus') was investigated by deoxyribonucleic acid hybridization. We confirm that all strains are highly related. Differences in the peptidoglycan type and the cytochrome pattern between S. mucilaginosus and members of Micrococcus, together with the results of 23 S ribosomal ribonucleic acid cistron similarity studies and the analysis of the 16S ribosomal ribonucleic acid support the exclusion of this species from the genus Micrococcus and justifies its reclassification as a member of a new genus, Stomatococcus (Bergan and Kocur, 1982). Phylogenetically, S. mucilaginosus represents an independent line of descent within a broad group of Gram-positive bacteria that contains arthrobacteria, micrococci, cellulomonads, brevibacteria and microbacteria.

Subtraction hybridization in microplates: an improved method to generate strain-specific PCR primers.

An improved subtraction hybridization technique was developed and evaluated. The hybridization is performed in a microplate with the subtractor-DNA immobilized in the plate while the probe-DNA is in solution. After hybridization the probe-specific DNA can easily be removed from the microwell and submitted to further analysis. This new technique has been successfully applied to generate several strain-specific PCR-primers for Lactococcus lactis subsp. lactis, Pediococcus spec., Saccharomyces spec. and Listeria monocytogenes.