16S-23S rDNA intergenic spacer region polymorphism of Lactococcus garvieae,Lactococcus raffinolactis and Lactococcus lactis as revealed by PCR and nucleotide sequence analysis

The intergenic spacer region (ISR) between the 16S and 23S rRNA genes was tested as a tool for differentiating lactococci commonly isolated in a dairy environment. 17 reference strains, representing 11 different species belonging to the genera Lactococcus, Streptococcus, Lactobacillus, Enterococcus and Leuconostoc, and 127 wild streptococcal strains isolated during the whole fermentation process of "Fior di Latte" cheese were analyzed. After 16S-23S rDNA ISR amplification by PCR, species or genus-specific patterns were obtained for most of the reference strains tested. Moreover, results obtained after nucleotide analysis show that the 16S-23S rDNA ISR sequences vary greatly, in size and sequence, among Lactococcus garvieae, Lactococcus raffinolactis, Lactococcus lactis as well as other streptococci from dairy environments. Because of the high degree of inter-specific polymorphism observed, 16S-23S rDNA ISR can be considered a good potential target for selecting species-specific molecular assays, such as PCR primer or probes, for a rapid and extremely reliable differentiation of dairy lactococcal isolates.     

Evaluation of a plasmid-based 16S-23S rDNA intergenic spacer region array for analysis of microbial diversity in industrial wastewater

A plasmid-based 16S-23S rDNA intergenic spacer region (ISR) array was developed and optimized for analysis of microbial diversity within complex environmental samples. Plasmid probes with 16S-23S rDNA ISR inserts (800-1500 bp) from industrial wastewater treatment plant (WWTP) microorganisms were arrayed onto glass slides. Hybridization of fluorescently labeled target sequences from two clones from the ISR WWTP library to arrayed probes showed that there was a good linear relationship between hybridization intensity and ISR similarity (r(2)=0.82). Hybridization was highly specific (average background from arrayed probes with less than 80% similarity in ISR sequence was less than 7%). Strong fluorescence intensity corresponded to near-perfect match clones (99% or greater similarity in ISR sequence). A majority of probes (79%) showed no background hybridization. However, weak background (less than 50% for arrayed probes with 90% and 95% similarity in the 16S rRNA genes) was observed from closely related microorganisms. Background fluorescence from the negative control (plasmid vector with no insert) was similar to water and dimethyl sulfoxide (DMSO)-negative controls. Hybridization using fluorescently labeled ISR sequences from a mixed community sample produced strong fluorescent signals with no background from negative controls. A Cy5-labeled reference standard, part of the vector and present in every spotted probe, was used to normalize hybridization values. These results indicate that arrayed plasmid containing ISR probe insert sequences provides specificity and sensitivity for microbial community analysis in a high-throughput array format.     

Cloning and sequencing of 16S rDNA and 16S-23S rDNA internal spacer region (ISR) from urease-positive thermophilic Campylobacter (UPTC)

AIMS: To clone and sequence the 16S rDNA and 16S-23S rDNA internal spacer region (ISR) from urease-positive thermophilic Campylobacter (UPTC). METHODS AND RESULTS: The primer sets for 16S rDNA and 16S-23S rDNA ISR amplified almost the full length of 16S rDNA and 16S-23S rDNA ISR. About 1500 bp for 16S rDNA and about 720 bp for 16S-23S rDNA ISR of the rrn operon of four strains of UPTC were identified after molecular cloning and sequencing. CONCLUSIONS: The four strains and CCUG18267 of UPTC showed approximately 99% sequence homology of 16S rDNA to each other, 96-97% to Camp. coli, 97-98% to Camp. jejuni and 97-98% to Camp. lari. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, the nucleotide sequence of 16S-23S rDNA ISR of UPTC has been analysed. The sequence of ISR was almost identical among the four strains of UPTC. It is interesting that the UPTC intercistronic tRNAs demonstrated an order of tRNA of 5'-16S-tRNAAla-tRNAIle-23S-3' in the organisms.     

Structural analysis and genetic variation of the 16S-23S rDNA internal spacer region from Micrococcus luteus strains

AIMS: To clone and sequence the 16S-23S ribosomal DNA (rDNA) internal spacer region (ISR) from Micrococcus luteus. METHODS AND RESULTS: The primer pair for 16S-23S rDNA ISR amplified a fragment of about 850 bp in length for two strains, JCM3347 and JCM3348 and a fragment of about 790 bp for a strain, ATCC9341. After sequencing the ISRs were identified by the comparison of the ISRs and the flanking regions of ISR. CONCLUSIONS: Although the sequence difference of the ISR occurred at only one position between the two JCM strains, the highly variable length (440 and 370 bp) and sequence similarity (about 40%) were demonstrated between the ISRs of the two JCM strains and a ATCC strain. SIGNIFICANCE AND IMPACT OF THE STUDY: A CCTCCT sequence was first detected at the 3'-end of the 16S rDNA of the three strains. Moreover, highly similar sequence to the 21-bp region containing a putative rRNA processing site was observed in the ISR of the three strains. Interestingly, no intercistronic tRNAs were demonstrated in the ISRs from the three strains.     

Candidatus "Scalindua brodae", sp. nov., Candidatus "Scalindua wagneri", sp. nov., two new species of anaerobic ammonium oxidizing bacteria

Anaerobic ammonium oxidation (anammox) is both a promising process in wastewater treatment and a long overlooked microbial physiology that can contribute significantly to biological nitrogen cycling in the world's oceans. Anammox is mediated by a monophyletic group of bacteria that branches deeply in the Planctomycetales. Here we describe a new genus and species of anaerobic ammonium oxidizing planctomycetes, discovered in a wastewater treatment plant (wwtp) treating landfill leachate in Pitsea, UK. The biomass from this wwtp showed high anammox activity (5.0 +/- 0.5 nmol/mg protein/min) and produced hydrazine from hydroxylamine, one of the unique features of anammox bacteria. Eight new planctomycete 16S rRNA gene sequences were present in the 16S rRNA gene clone library generated from the biomass. Four of these were affiliated to known anammox 16S rRNA gene sequences, but branched much closer to the root of the planctomycete line of descent. Fluorescence in situ hybridization (FISH) with oligonucleotide probes specific for these new sequences showed that two species (belonging to the same genus) together made up > 99% of the planctomycete population which constituted 20% of the total microbial community. The identification of these organisms as typical anammox bacteria was confirmed with electron microscopy and lipid analysis. The new species, provisionally named Candidatus "Scalindua brodae" and "Scalindua wagneri" considerably extend the biodiversity of the anammox lineage on the 16S rRNA gene level, but otherwise resemble known anammox bacteria. Simultaneously, another new species of the same genus, Candidatus "Scalindua sorokinii", was detected in the water column of the Black Sea, making this genus the most widespread of all anammox bacteria described so far.     

Molecular evidence for genus level diversity of bacteria capable of catalyzing anaerobic ammonium oxidation

Recently, a bacterium capable to oxidize ammonium anaerobically at a high rate was identified as novel member of the Planctomycetales (Strous, M., Fuersi, J. A., Kramer, E. H. M., Logemann, S., Muyzer, G., van de Pas-Schoonen, K. T., Webb, R. I., Kufnen, J. G., and Jetten, M. S. M.: Nature 400, 446-449, 1999). Here we investigated the microbial community structure of a trickling filter biofilm with a high anaerobic ammonium oxidation activity. Fluorescence in situ hybridization (FISH) with a set of nine probes designed for specific identification of the recently described anaerobic ammonium oxidizer demonstrated that only one probe hybridized to bacteria within the biofilm. For phylogenetic characterization of putative biofilm anaerobic ammonium oxidizers a full-cycle 16S rDNA approach was performed by using a Planctomycetales-specific forward primer for PCR amplification. Of the twenty-five 16S rDNA fragments (1364 bp in length) amplified from the biofilm, nine were affiliated to the Planctomycetales. Comparative analysis showed that these sequences were more than 98.9% similar to each other but only distantly related to the previously recognized anaerobic ammonium oxidizer (below 91% similarity) and all other organisms represented in public 16S rRNA databases (similarities of below 79%). The retrieved sequences and the previously recognized anaerobic ammonium oxidizer represent two well-separated groups of a deep-branching lineage within the Planctomycetales. Quantitative FISH analysis with a newly designed specific probe showed that the novel bacterium, provisionally classified as "Candidatus Kuenenia stuttgartiensis" constituted the dominant fraction of the biofilm bacteria. In situ probing revealed that ammonia-oxidizing bacteria of the beta-subclass of Proteobacteria were also present, albeit in significant smaller amounts, within the anoxic biofilm. Comparative sequence analysis of a stretch of the gene encoding ammonia-monooxygenase (amoA) demonstrated the occurrence of the DNA of at least three different populations of beta-subclass ammonia oxidizers within the biofilm.     

Inter- and intraspecies variations of the 16S-23S rDNA intergenic spacer region of various streptococcal species

The 16S-23S rDNA intergenic spacer regions (ISR) of different streptococcal species and subspecies were amplified with primers derived from the highly conserved flanking regions of the 16S rRNA and 23S rRNA genes. The single sized amplicons showed a uniform pattern for S. agalactiae, S. dysgalactiae subsp. dysgalactiae (serogroup C), S. dysgalactiae subsp. equisimilis (serogroup G), S. dysgalactiae subsp. dysgalactiae (serogroup L), S. canis, S. phocae, S. uberis, S. parauberis, S. pyogenes and S. equi subsp. equi, respectively. The amplicons of S. equi subsp. zooepidemicus, S. porcinus and S. suis appeared with 3, 5 and 3 different sizes, respectively. ISR of selected strains of each species or subspecies investigated were sequenced and multiple aligned. This allowed a separation of ISR into regions, with 7 regions for S. agalactiae, S. dysgalactiae subsp. dysgalactiae (serogroup C), S. dysgalactiae subsp. equisimilis (serogroup G), S. dysgalactiae subsp. dysgalactiae (serogroup L), S. canis, S. phocae, S. pyogenes and S. suis, 8 regions for S. uberis and S. parauberis and mostly 9 regions for S. equi subsp. equi, S. equi subsp. zooepidemicus and S. porcinus. Region 4, encoding the transfer RNA for alanine (tRNA(Ala)), was present and identical for all isolates investigated. The size and sequence of ISR appears to be a unique marker for streptococci of various species and subspecies and could be used for bacterial identification. In addition the size and sequence variations of ISR of S. equi subsp. zooepidemicus, S. porcinus and S. suis allows a molecular typing of isolates of these species possibly useful in epidemiological aspects.     

Fast protocols for the 5S rDNA and ITS-2 based identification of Oenococcus oeni

To identify specific marker sequences for the rapid identification of Oenococcus oeni, we sequenced the 23S-5S internal transcribed spacer (ITS-2) region and the 5S rDNA of five different O. oeni strains and three phylogenetically related lactic acid bacteria (LAB). Comparative analysis revealed 100% identity among the ITS-2 region of the O. oeni strains and remarkable differences in length and sequence compared to related LAB. These results enabled us to develop a primer set for a rapid PCR-identification of O. oeni within three hours. Moreover, the comparison of the 5S rDNA sequences and the highly conserved secondary structure provided the template for the design of three fluorescence-labeled specific oligonucleotides for fluorescence in situ hybridization (FISH). These probes are partial complementary to each other. This feature promotes the accessibility to the target sequence within the ribosome and enhances the fluorescence signal. For the rapid identification of Oenococci both the 5S rRNA gene and the ITS-2 region are useful targets.     

The rRNA operon from Zea mays chloroplasts: nucleotide sequence of 23S rDNA and its homology with E.coli 23S rDNA.

The nucleotide sequence of 23S rDNA from Zea mays chloroplasts has been determined. Alignment with 23S rDNA from E.coli reveals 71 percent homology when maize 4.5S rDNA is included as an equivalent of the 3' end of E.coli 23S rDNA. Among the conserved sequences are sites for base modification. Chloramphenicol sensitivity and ribosomal subunit interaction. A proposal for the base pairs formed between 16S and 23S rRNAs during the 30S/50S subunit interaction is presented. The alignment of maize 23S rDNA with that of E.coli reveals three small insertion sequences of 25, 65 and 78 base pairs, whereas maize 16S rDNA shows only deletions when compared with the E.coli species.     

Novel bacterial endosymbionts of Acanthamoeba spp. related to the Paramecium caudatum symbiont Caedibacter caryophilus

Acanthamoebae are increasingly being recognized as hosts for obligate bacterial endosymbionts, most of which are presently uncharacterized. In this study, the phylogeny of three Gram-negative, rod-shaped endosymbionts and their Acanthamoeba host cells was analysed by the rRNA approach. Comparative analyses of 16S rDNA sequences retrieved from amoebic cell lysates revealed that the endosymbionts of Acanthamoeba polyphaga HN-3, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39 are related to the Paramecium caudatum endosymbionts Caedibacter caryophilus, Holospora elegans a n d Holospora obtusa . With overall 16S rRNA sequence similarities to their closest relative, C. caryophilus , of between 87% and 93%, these endosymbionts represent three distinct new species. In situ hybridization with fluorescently labelled endosymbiont-specific 16S rRNA-targeted probes demonstrated that the retrieved 16S rDNA sequences originated from the endosymbionts and confirmed their intracellular localization. We propose to classify provisionally the endosymbiont of Acanthamoeba polyphaga HN-3 as ' Candidatus Caedibacter acanthamoebae', the endosymbiont of Acanthamoeba sp. strain UWC9 as ' Candidatus Paracaedibacter acanthamoebae' and the endosymbiont of Acanthamoeba sp. strain UWE39 as ' Candidatus Paracaedibacter symbiosus'. The phylogeny of the Acanthamoeba host cells was analysed by comparative sequence analyses of their 18S rRNA. Although Acanthamoeba polyphaga HN-3 clearly groups together with most of the known Acanthamoeba isolates (18S rRNA sequence type 4), Acanthamoeba sp. UWC9 and UWE39 exhibit < 92% 18S rRNA sequence similarity to each other and to other Acanthamoeba isolates. Therefore, we propose two new sequence types (T13 and T14) within the genus Acanthamoeba containing, respectively, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39.     

A one-step reaction for the rapid identification of Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti using oligonucleotide primers designed from the 16S-23S rRNA intergenic sequences

Aims:  Species-specific primers targeting the 16S–23S ribosomal DNA (rDNA) intergenic spacer region (ISR) were designed to rapidly discriminate between Lactobacillus mindensis , Lactobacillus panis , Lactobacillus paralimentarius , Lactobacillus pontis and Lactobacillus frumenti species recently isolated from French sourdough.
Methods and Results:  The 16S–23S ISRs were amplified using primers 16S/p2 and 23S/p7, which anneal to positions 1388–1406 of the 16S rRNA gene and to positions 207–189 of the 23S rRNA gene respectively, Escherichia coli numbering (GenBank accession number V00331 ). Clone libraries of the resulting amplicons were constructed using a pCR2·1 TA cloning kit and sequenced. Species-specific primers were designed based on the sequences obtained and were used to amplify the 16S–23S ISR in the Lactobacillus species considered. For all of them, two PCR amplicons, designated as small ISR (S-ISR) and large ISR (L-ISR), were obtained. The L-ISR is composed of the corresponding S-ISR, interrupted by a sequence containing tRNA^Ile and tRNA^Ala genes. Based on these sequences, species-specific primers were designed and proved to identify accurately the species considered among 30 reference Lactobacillus species tested.
Conclusions:  Designed species-specific primers enable a rapid and accurate identification of L. mindensis , L. paralimentarius , L. panis , L. pontis and L. frumenti species among other lactobacilli.
Significance and Impact of the Study:  The proposed method provides a powerful and convenient means of rapidly identifying some sourdough lactobacilli, which could be of help in large starter culture surveys.     

16S to 23S rDNA spacer fragment length polymorphism of Aeromonas sp

We analyzed polymorphism of the PCR-amplified 16S-23S rDNA spacer of Aeromonas species. A total of 69 isolates representing 18 DNA hybridization groups were used in this study. The analysis of PCR products of 16S-23S rDNA spacers revealed patterns consisting of two to eight DNA fragments. The fragment sizes ranged from 730 to 1050 bp. DNA patterns revealed a considerable genetic diversity between species and within a species. When a procedure to eliminate heteroduplex formation was performed, the number of bands was reduced to 2-5. Nevertheless the homoduplex ISR (intergenic spacer region) patterns obtained were not useful for species distinguishing.     

Applicability of the 16S–23S rDNA internal spacer for PCR detection of the phytostimulatory PGPR inoculant Azospirillum lipoferum CRT1 in field soil

Aims:  To assess the applicability of the 16S–23S rDNA internal spacer regions (ISR) as targets for PCR detection of Azospirillum ssp. and the phytostimulatory plant growth-promoting rhizobacteria seed inoculant Azospirillum lipoferum CRT1 in soil.
Methods and Results:  Primer sets were designed after sequence analysis of the ISR of A. lipoferum CRT1 and Azospirillum brasilense Sp245. The primers fAZO/rAZO targeting the Azospirillum genus successfully yielded PCR amplicons (400–550 bp) from Azospirillum strains but also from certain non- Azospirillum strains in vitro , therefore they were not appropriate to monitor indigenous Azospirillum soil populations. The primers fCRT1/rCRT1 targeting A. lipoferum CRT1 generated a single 249-bp PCR product but could also amplify other strains from the same species. However, with DNA extracts from the rhizosphere of field-grown maize, both fAZO/rAZO and fCRT1/rCRT1 primer sets could be used to evidence strain CRT1 in inoculated plants by nested PCR, after a first ISR amplification with universal ribosomal primers. In soil, a 7-log dynamic range of detection (10²–10⁸ CFU g⁻¹ soil) was obtained.
Conclusions:  The PCR primers targeting 16S–23S rDNA ISR sequences enabled detection of the inoculant A. lipoferum CRT1 in field soil.
Significance and Impact of the Study:  Convenient methods to monitor Azospirillum phytostimulators in the soil are lacking. The PCR protocols designed based on ISR sequences will be useful for detection of the crop inoculant A. lipoferum CRT1 under field conditions.     

The role of recombination and mutation in 16S-23S rDNA spacer rearrangements

The intragenomic heterogeneity of the bacterial intergenic (16S-23S rDNA) spacer region (ISR) was analysed from the following species in which sequences for the complete rRNA operon (rrn) set have been determined (rrn number): Enterococcus faecalis (6) and E. faecium (6), Bacillus subtilis (10), Staphylococcus aureus (9), Vibrio cholerae (4), Haemophilus influenzae (6) and Escherichia coli (7). It was found that some spacer sequence blocks were highly conserved between operons of a genome, whereas the presence of others was variable. When these variations were analysed using the program PLATO and partial likelihood phylogenies determined by DNAml for each operon set, three regions showed significant (Z>3.3) spatial variation [Region I was 78-184 nt long (2.14.4) possibly due to recombination or selection. Within Region I, there was sequence block variation in all operon sets [some operons contained tRNA genes (tRNAala, tRNAile or tRNAglu), whereas others had sequence blocks such as VS2 (S. aureus) or rsl (E. coli)]. Q Analysis of the ISR sequence from E. faecalis and E. faecium showed that there was more interspecies than intraspecies variation (both in DNA sequence and in the presence or absence of blocks). Dot matrix analysis of the sequence blocks in the nine rrn ISRs from S. aureus showed that there was significant homology between VS2 and VS5/VS6. Furthermore, repeat motifs with only A or T were present in higher copy numbers in VS5/VS6 than in VS2. Since these sequence blocks (VS2 and VS5-VS6) are related, intragenic evolution resulting in AT expansion may have occurred between these two regions. A model is proposed that postulates a role for recombination and AT-expansion in intra-genomic ISR variations. This process may represent a general mechanism of concerted evolution for bacterial ISR rearrangements.     

Cotranscription and processing of 23S, 4.5S and 5S rRNA in chloroplasts from Zea mays

The termini of rRNA processing intermediates and of mature rRNA species encoded by the 3' terminal region of 23S rDNA, by 4.5S rDNA, by the 5' terminal region of 5S rDNA and by the 23S/4.5S/5S intergenic regions from Zea mays chloroplast DNA were determined by using total RNA isolated from maize chloroplasts and 32P-labelled rDNA restriction fragments of these regions for nuclease S1 and primer extension mapping. Several processing sites detectable by both 3' and 5' terminally labelled probes could be identified and correlated to the secondary structure for the 23S/4.5S intergenic region. The complete 4.5S/5S intergenic region can be reverse transcribed and a common processing site for maturation of 4.5S and 5S rRNA close to the 3' end of 4.5S rRNA was detected. It is therefore concluded that 23S, 4.5S and 5S rRNA are cotranscribed.     

Microbial Composition and Structure of a Rotating Biological Contactor Biofilm Treating Ammonium-Rich Wastewater without Organic Carbon

High nitrogen losses were observed in a rotating biological contactor (RBC) treating ammonium-rich (up to 500 mg NH4(+)-N/L) but organic-carbon-poor leachate from a hazardous waste landfill in K?lliken, Switzerland. The composition and spatial structure of the microbial community in the biofilm on the RBC was analyzed with specific attention for the presence of aerobic ammonium and nitrite oxidizing bacteria and anaerobic ammonium oxidizers. Anaerobic ammonium oxidation (anammox) involves the oxidation of ammonium with nitrite to N2. First the diversity of the biofilm community was determined from sequencing cloned PCR-amplified 16S rDNA fragments. This revealed the presence of a number of very unusual 16S rDNA sequences, but very few sequences related to known ammonium or nitrite oxidizing bacteria. From analysis of biofilm samples by fluorescence in situ hybridization with known phylogenetic probes and by dot-blot hybridization of the same probes to total RNA purified from biofilm samples, the main groups of microorganisms constituting the biofilm were found to be ammonium-oxidizing bacteria from the Nitrosomonas europaea/eutropha group, anaerobic ammonium-oxidizing bacteria of the "Candidatus Kuenenia stuttgartiensis" type, filamentous bacteria from the phylum Bacteroidetes, and nitrite-oxidizing bacteria from the genus Nitrospira. Aerobic and anaerobic ammonium-oxidizing bacteria were present in similar amounts of around 20 to 30% of the biomass, whereas members of the CFB phylum were present at around 7%. Nitrite oxidizing bacteria were only present in relatively low amounts (less than 5% determined with fluorescence in situ hybridization). Data from 16S rRNA dot-blot and in situ hybridization were not in all cases congruent. FISH analysis of thin-sliced and fixed biofilm samples clearly showed that the aerobic nitrifiers were located at the top of the biofilm in an extremely high density and in alternating clusters. Anammox bacteria were exclusively present in the lower half of the biofilm, whereas CFB-type filamentous bacteria were present throughout the biofilm. The structure and composition of these biofilms correlated very nicely with the proposed physiological functional separations in ammonium conversion.     

斜茎黄芪根瘤菌的16SrDNA和23SrDNAPCR—RFLP比较分析

在表型性状数值分析和ＡＦＬＰ指纹图谱分析的基础上,选取５４株斜茎黄芪根瘤菌的代表菌株及已知根瘤菌参比菌株,进行１６ＳｒＤＮＡ和２３ＳｒＤＮＡ的ＰＣＲ－ＲＦＬＰ比较分析。结果表明斜茎黄芪根瘤菌具有极大的系统发育多样性,分别具有２４个１６ＳｒＤＮＡ遗传图谱类型和２２个２３ＳｒＤＮＡ遗传图谱类型,１６ＳｒＤＮＡ与２３ＳｒＤＮＡＰＣＲ－ＲＦＬＰ聚类分析树状图谱有较好的一致性,但也存在一些差异。在对较大类群的划分上,它们的结果与表型性状数值分析结果有较好的一致性。将１６ＳｒＤＮＡ和２３ＳｒＤＮＡＰＣＲ－ＲＦＬＰ分析数据合并在一起进行分析时,得出２６个综合遗传图谱类型和１个综合聚类分析树状图谱。很明显,１６ＳｒＤＮＡ与２３ＳｒＤＮＡ的合并,能够得出更可靠的系统发育结论。     

Molecular characterization and physical mapping of two classes of 5S rDNA in the genomes of Gymnotus sylvius and G. inaequilabiatus (Gymnotiformes, Gymnotidae)

The nucleotide sequences of the 5S rRNA multigene family and their distribution across the karyotypes in 2 species of Gymnotiformes, genus Gymnotus (G. sylvius and G. inaequilabiatus) were investigated by means of fluorescence in situ hybridization (FISH). The results showed the existence of 2 distinct classes of 5S rDNA sequences in both species: class I and class II. A high conservative pattern of the codifying region of the 5S rRNA gene was identified, contrasting with significant alterations detected in the nontranscribed spacer (NTS). The presence of TATA-like sequences along the NTS of both species was an expected occurrence, since such sequences have been associated with the regulation of the gene expression. FISH using 5S rDNA class I and class II probes revealed that both gene classes were collocated in the same chromosome pair in the genome of G. sylvius, while in that of G. inaequilabiatus, class II appeared more disperse than class I.     

Enrichment and characterization of an anammox bacterium from a rotating biological contactor treating ammonium-rich leachate

Anaerobic ammonium oxidation with nitrite to N2 (anammox) is a recently discovered microbial reaction with interesting potential for nitrogen removal from wastewater. We enriched an anammox culture from a rotating disk contactor (near K?lliken, Switzerland) that was used to treat ammonium-rich leachate with low organic carbon content. This enrichment led to a relative population size of 88% anammox bacteria. The microorganism carrying out the anammox reaction was identified by analysis of the 16S rDNA sequence and by fluorescence in situ hybridization (FISH) with 16S-rRNA-targeting probes. The percentage sequence identity between the 16S rDNA sequences of the K?lliken anammox organism and the archetype anammox strain Candidatus Brocadia anammoxidans was 90.9%, but between 98.5 and 98.9% with Candidatus Kuenenia stuttgartiensis, an organism identified in biofilms by molecular methods. The K?lliken culture catalyzed the anaerobic oxidation of ammonium with nitrite in a manner seemingly identical to that of Candidatus B. anammoxidans, but exhibited higher tolerance to phosphate (up to 20 mM) and to nitrite (up to 13 mM) and was active at lower cell densities. Anammox activity was observed only between pH 6.5 and 9, with an optimum at pH 8 and a temperature optimum at 37 degrees C. Hydroxylamine and hydrazine, which are intermediates of the anammox reaction of Candidatus B. anammoxidans, were utilized by the K?lliken organisms, and approximately 15% of the nitrite utilized during autotrophic growth was converted to nitrate. Electron microscopy showed a protein-rich region in the center of the cells surrounded by a doughnut-shaped region containing ribosomes and DNA. This doughnut-shape region was observed with FISH as having a higher fluorescence intensity. Similar to Candidatus B. anammoxidans, the K?lliken anammox organism typically formed homogenous clusters containing up to several hundred cells within an extracellular matrix.     

Typing of clinical and environmental Aeromonas veronii strains based on the 16S-23S rDNA spacers

Genetic relationships of Aeromonas veronii strains isolated from human and environmental sources were investigated by restriction fragment length polymorphism (RFLP) of the polymerase chain reaction-amplified intergenic spacer region (ISR) flanked by the 16S and 23S rRNA genes. When using endonucleases AluI, HinfI and CfoI the 16S-23S rDNA-RFLP patterns showed considerable overall similarity, although most strains yielded specific profiles. Several intra-specific lines of descent comprised clinical strains linked to isolates from environmental sources. Strains having identical patterns may be individuals derived from highly similar, if not the same, microorganism. Results suggest that the ISR sequence-based method can be used to demonstrate colonization of a public water supply with a particular microorganism. In addition it could be very useful for tracing recurrent episodes of diarrhea and Aeromonas infection outbreaks.