Construction of a Cloning System for the Mass Production of a Virus-Binding Protein Specific for Poliovirus Type 1

In our previous study, virus-binding proteins (VBPs) demonstrating the ability to strongly bind poliovirus type 1 (PV1) were recovered from a bacterial culture derived from activated sludge. The isolated VBPs would be useful as viral adsorbents for water and wastewater treatments. The VBP gene of activated sludge bacteria was isolated, and the cloning system of the VBP was established. The isolation of the VBP gene from DNA libraries for activated sludge bacteria was achieved with the colony hybridization technique. The sequence of the VBP gene consisted of 807 nucleotides encoding 268 amino acids. Fifteen amino acid sequences were retrieved from 2,137,877 sequences by a homology search using the BLAST server at the National Center for Biotechnology Information. The protein encoded in the isolated genome was considered to be a newly discovered protein from activated sludge culture, because any sequences in protein databases were not perfectly matched with the sequence of the VBP. It was confirmed that Escherichia coli BL21 transformed by pRSET carrying the isolated VBP gene could extensively produce the VBP clones. Enzyme-linked immunosorbent assay (ELISA) revealed that the VBP clone exhibited the binding ability with intact particles of PV1. The equilibrium binding constant between PV1 and VBP in the ELISA well was estimated to be 2.1 × 10⁷ (M⁻¹), which also indicated that the VBP clones have a high affinity with the PV1 particle. The VBP cloning system developed in this study would make it possible to produce a mass volume of VBPs and to utilize them as a new material of the specific adsorbent in several technologies, including virus removal, concentration, and detection.     

Virus-binding proteins recovered from bacterial culture derived from activated sludge by affinity chromatography assay using a viral capsid peptide

The contamination of water environments by pathogenic viruses has raised concerns about outbreaks of viral infectious diseases in our society. Because conventional water and wastewater treatment systems are not effective enough to inactivate or remove pathogenic viruses, a new technology for virus removal needs to be developed. In this study, the virus-binding proteins (VBPs) in a bacterial culture derived from activated sludge were successfully recovered. The recovery of VBPs was achieved by applying extracted crude proteins from a bacterial culture to an affinity column in which a custom-made peptide of capsid protein from the poliovirus type 1 (PV1) Mahoney strain (H(2)N-DNPASTTNKDKL-COOH) was immobilized as a ligand. VBPs exhibited the ability to adsorb infectious particles of PV1 Sabin 1 as determined by enzyme-linked immunosorbent assay. The evaluation of surface charges of VBPs with ion-exchange chromatography found that a majority of VBP molecules had a net negative charge under the conditions of affinity chromatography. On the other hand, a calculated isoelectric point implied that the viral peptide in the affinity column was also charged negatively. As a result, the adsorption of the VBPs to the viral peptide in the affinity column occurred with a strong attractive force that was able to overcome the electrostatic repulsive force. Two-dimensional electrophoresis revealed that the isolated VBPs include a number of proteins, and their molecular masses were widely distributed but smaller than 100 kDa. Amino acid sequences of N termini of five VBPs were determined. Homology searches for the N termini against all protein sequences in the National Center for Biotechnology Information (NCBI) database showed that the isolated VBPs in this study were newly discovered proteins. These VBPs that originated with bacteria in activated sludge might be stable, because they are existing in the environment of wastewater treatments. Therefore, a virus removal technology utilizing VBPs as viral adsorbents can be developed, since it is possible to replicate VBPs by protein cloning techniques.     

Virus-Binding Proteins Recovered from Bacterial Culture Derived from Activated Sludge by Affinity Chromatography Assay Using a Viral Capsid Peptide

The contamination of water environments by pathogenic viruses has raised concerns about outbreaks of viral infectious diseases in our society. Because conventional water and wastewater treatment systems are not effective enough to inactivate or remove pathogenic viruses, a new technology for virus removal needs to be developed. In this study, the virus-binding proteins (VBPs) in a bacterial culture derived from activated sludge were successfully recovered. The recovery of VBPs was achieved by applying extracted crude proteins from a bacterial culture to an affinity column in which a custom-made peptide of capsid protein from the poliovirus type 1 (PV1) Mahoney strain (H₂N-DNPASTTNKDKL-COOH) was immobilized as a ligand. VBPs exhibited the ability to adsorb infectious particles of PV1 Sabin 1 as determined by enzyme-linked immunosorbent assay. The evaluation of surface charges of VBPs with ion-exchange chromatography found that a majority of VBP molecules had a net negative charge under the conditions of affinity chromatography. On the other hand, a calculated isoelectric point implied that the viral peptide in the affinity column was also charged negatively. As a result, the adsorption of the VBPs to the viral peptide in the affinity column occurred with a strong attractive force that was able to overcome the electrostatic repulsive force. Two-dimensional electrophoresis revealed that the isolated VBPs include a number of proteins, and their molecular masses were widely distributed but smaller than 100 kDa. Amino acid sequences of N termini of five VBPs were determined. Homology searches for the N termini against all protein sequences in the National Center for Biotechnology Information (NCBI) database showed that the isolated VBPs in this study were newly discovered proteins. These VBPs that originated with bacteria in activated sludge might be stable, because they are existing in the environment of wastewater treatments. Therefore, a virus removal technology utilizing VBPs as viral adsorbents can be developed, since it is possible to replicate VBPs by protein cloning techniques.     

Nucleotide sequence of the structural gene, tcpA, for a major pilin subunit of Vibrio cholerae

The toxin co-regulated pilus (Tcp) of Vibrio cholerae appears to be a major protective antigen. By cosmid cloning we have isolated a number of clones capable of converting Tcp- El Tor strains of V. cholerae to Tcp+. A synthetic oligodeoxyribonucleotide probe based upon the N-terminal amino acid sequence of TcpA, has been used to localize the structural gene within the cosmid clones. Using suitable subclones, the nucleotide sequence of the tcpA gene has been determined. The gene encodes a 23.3-kDa pre-protein which in its mature form has a size of 20.3 kDa. The N-terminal leader peptide or signal sequence is atypical and does not conform with the usual rules of such sequences. The TcpA protein shows some similarities to the major pilins of the methylated phenylalanine type or type-4 pili from other bacteria; however, it is sufficiently different that it may represent a new class.     

Agrobacterium VirD2-binding protein is involved in tumorigenesis and redundantly encoded in conjugative transfer gene clusters

Agrobacterium tumefaciens can transfer oncogenic T-DNA into plant cells; T-DNA transfer is mechanistically similar to a conjugation process. VirD2 is the pilot protein that guides the transfer, because it is covalently associated with single-stranded T-DNA to form the transfer substrate T-complex. We used the VirD2 protein as an affinity ligand to isolate VirD2-binding proteins (VBPs). By pull-down assays and peptide-mass-fingerprint matching, we identified an A. tumefaciens protein designated VBP1 that could bind VirD2 directly. Genome-wide sequence analysis showed that A. tumefaciens has two additional genes encoding proteins highly similar to VBP1, designated vbp2 and vbp3. Like VBP1, both VBP2 and VBP3 also could bind VirD2; all three VBPs contain a putative nucleotidyltransferase motif. Mutational analysis of vbp demonstrated that the three vbp genes could functionally complement each other. Consequently, only inactivation of all three vbp genes highly attenuated the bacterial ability to cause tumors on plants. Although vbp1 is harbored on the megaplasmid pAtC58, vbp2 and vbp3 reside on the linear chromosome. The vbp genes are clustered with conjugative transfer genes, suggesting linkage between the conjugation and virulence factor. The three VBPs appear to contain C-terminal positively charged residues, often present in the transfer substrate proteins of type IV secretion systems. Inactivation of the three vbp genes did not affect the T-strand production. Our data indicate that VBP is a newly identified virulence factor that may affect the transfer process subsequent to T-DNA production.     

东亚钳蝎神经毒素在大肠杆菌中的表达

以山西风陵渡东亚钳蝎（ＢｕｔｈｕｓｍａｒｔｅｎｓｉＫａｒｓｃｈ）的尾腺总ＲＮＡ为模板,根据已知的蝎神经毒素保守氨基酸序列设计引物,利用ＰＣＲ技术,扩增并克隆了两个蝎神经毒素基因．序列分析表明,由两个基因导出的氨基酸序列（ＢｍＫＭｍ１和ＢｍＫＭｍ２）与已知的蝎神经毒素ＢｍＫⅠ、ＢｍＫⅡ、ＢｍＫⅢ、ＢｍＫＭ１、ＢｍＫＭ９有很高的同源性．将ＢｍＫＭｍ２基因重组到大肠杆菌分泌型表达载体ｐＥｘＳｅｃ１中进行表达．ＳＤＳ－ＰＡＧＥ证明表达产物被分泌到细胞周间质及培养液中．经ＩｇＧ－Ｓｅｐｈａｒｏｓｅ纯化后的蛋白质注射小白鼠表明表达产物有生物学活性     

Mass isolation of cuticle protein cDNAs from wing discs of Bombyx mori and their characterizations

Multiple cloning of cuticle protein genes was performed by sequencing of cDNAs randomly selected from a cDNA library of wing discs just before pupation, and nine different cuticular protein genes were identified. Thirty-one clones of a cuticle protein gene were identified from the 1050 randomly sequenced clones; about 3% were cuticle protein genes in the W3-stage wing disc cDNA library. The sequence diversity of the deduced amino acid sequences of isolated Bombyx cuticle genes was examined along with the expression profiles. The deduced amino acid sequences of the nine cuticle protein genes contained a putative signal peptide at the N-terminal region and a very conserved hydrophilic region known as the R and R motif. The developmental expression of cuticle genes was classified into two types: pupation (five clones were expressed only around pupation) and pupation and mid-pupal (four clones were expressed around this stage). All the isolated genes were expressed in the head, thoracic, and abdominal regions of the epidermis at different levels around pupation, but no expression was observed in the epidermis at the fourth molting stage.     

Quantification of Hyphomicrobium populations in activated sludge from an industrial wastewater treatment system as determined by 16S rRNA analysis

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869(T) in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.     

Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli.

The gene (xynA) encoding a surface-exposed, S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 was cloned and expressed in Escherichia coli. A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria. This PCR product was used as a probe in Southern hybridizations to identify a 4.6-kb EcoRI fragment containing the complete xynA gene. This fragment was cloned into E. coli, and recombinant clones expressed significant levels of xylanase activity. The purified recombinant protein had an estimated molecular mass (150 kDa), temperature maximum (80 degrees C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485. The entire insert was sequenced and analysis revealed a 4,044-bp open reading frame encoding a protein containing 1,348 amino acid residues (estimated molecular mass of 148 kDa).xynA was preceded by a putative promoter at -35 (TTAAT) and -10 (TATATT) and a potential ribosome binding site (AGGGAG) and was expressed constitutively in E. coli. The deduced amino acid sequence showed 30 to 96% similarity to sequences of family F beta-glycanases. A putative 32-amino-acid signal peptide was identified, and the C-terminal end of the protein contained three repeating sequences 59, 64, and 57 amino acids) that showed 46 to 68% similarity to repeating sequences at the N-terminal end of S-layer and S-layer-associated proteins from other gram-positive bacteria. These repeats could permit an interaction of the enzyme with the S-layer and tether it to the cell surface.     

Sequence and expression of human protein kinase C-epsilon.

Two human homologues of protein kinase C-epsilon (E1 and E2) were isolated from two distinct cDNA libraries. Sequence comparisons to PKC-epsilon cDNAs from several species indicated that each of these human epsilon clones contained cloning artifacts. Thus, a composite PKC-epsilon (E3) clone was derived from clones E1 and E2. Human PKC-epsilon (E3) has an overall sequence identity of 90-92% at the nucleotide level compared to the previously characterized mouse, rat and rabbit clones. At the amino acid level, the deduced human epsilon sequence shows a 98-99% identity with the mouse, rat and rabbit sequences. Expression of the human PKC-epsilon clone in Sf9 cells confirmed that the recombinant protein displayed protein kinase C activity and phorbol ester binding activity. The recombinant protein was also recognized by two distinct epsilon-specific polyclonal antibodies.     

Isolation of a Drosophila gene coding for a protein containing a novel phosphatidylserine-binding motif

To elucidate the molecular basis of the binding of proteins to the membrane phospholipid phosphatidylserine (PS), we characterized PS-binding peptides isolated from a phage display library. Amino acid sequences deduced from the nucleotide sequences of over 60 phage clones isolated revealed that there was no common primary structure among these peptides, but all peptides were rich in basic amino acid residues. In particular, 15 clones encoded peptides that contained contiguous arginine residues. Characterization of two such peptides in more detail showed that they bound to PS, and to a much lower extent to other phospholipids, including phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine. Unlike other Ca2+-dependent PS-binding proteins, these peptides did not require Ca2+ for binding to PS, and the addition of Ca2+ did not alter the phospholipid specificity. Substitution of one of the two RR sequences in one peptide by alanine had no effect, but that of both sequences completely abolished the activity. Furthermore, we identified a Drosophila gene coding for a presumed nuclear protein that shares an amino acid sequence, including a RR residue, with one of the two PS-binding peptides. This protein bound to PS partly depending on the presence of the RR residue. These results allowed us to conclude that an amino acid sequence including contiguous arginine residues is a novel motif that defines Ca2+-independent PS-binding activity.     

Immunological comparison of the in vitro and in vivo labeled victorin binding protein from susceptible oats

The fungus Cochliobolus victoriae causes victoria blight of oats and produces the host-specific toxin victorin. The reaction of oats to the fungus and its toxin is controlled by a single dominant gene whose product has been hypothesized to function as the site of action (receptor) of the toxin in susceptible oat genotypes. Previously, using a biologically active ¹²⁵I derivative of the toxin, we identified a 100 kilodalton victorin-binding protein (VBP) which binds victorin in a ligand-specific manner and binds in vivo only in susceptible oat genotypes. However, a VBP in both the susceptible and resistant oat genotypes was identified by in vitro binding experiments. One interpretation of the lack of genotype-specific binding in vitro is that the 100 kilodalton protein detected in vitro is not the same 100 kilodalton protein detected in vivo. To clarify the relationship between the 100 kilodalton protein(s) labeled in vivo and in vitro, we developed antisera to the in vitro-labeled VBP from the susceptible genotype and demonstrated that these preparations react with the in vivo-labeled VBP from the susceptible genotype. This finding coupled with previous observations strongly suggest that the VBP observed in vivo is the same protein detected in vitro. Furthermore, the results support our previous observations which suggest that the VBPs labeled in vitro in susceptible and resistant genotypes are closely related or identical.     

Density Gradient Study of Victorin-Binding Proteins in Oat (Avena sativa) Cells

Victorin-binding proteins (VBPs) in oat (Avena sativa) cells were identified using native victorin and anti-victorin polyclonal antibodies. Homogenates of oat tissues were fractionated in continuous or discontinuous sucrose density gradients or with an aqueous two-phase method, and covalent binding sites of victorin were detected by western blotting. In a 20 to 45% (w/w) sucrose continuous density gradient, the 100-kD VBP was located in fractions of 37 to 44% sucrose, with a peak at 39% sucrose. Based on marker enzyme assays, plasma membranes peaked at 39 to 41% sucrose, mitochondria peaked at 41%, but Golgi and endoplasmic reticulum were in lower density fractions, peaking at 28 to 29% and 22 to 24% sucrose, respectively. The 100-kD VBP was not found in plasma membranes purified by the aqueous two-phase method or in mitochondria purified by discontinuous density gradient centrifugation. Victorin binding to 65- and 45-kD proteins was detected in all fractions in the continuous sucrose density gradients. The 65- and 45-kD proteins were both detected in purified plasma membranes, but only the 65-kD protein was detected in purified mitochondria. The subcellular location of VBPs was the same in sensitive and resistant oat cells.     

Isolation of cDNAs encoding for two distinct isoforms of the TATA-Box binding proteins from common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

The TATA-box binding protein (TBP) is one of the 4 DNA-binding proteins that has been shown to associate with the proximal promoter region (−295) of the gene for bean seed storage protein phaseolin. The −295 promoter is essential for spatial and temporal control of the phaseolin gene expression. We designed a pair of degenerated primers based on the highly conserved sequence of the carboxyl-terminal domain of yeast TBP and used PCR to amplify the corresponding sequence from the bean cDNA. By using the amplified fragment as a probe, we screened a cDNA library derived from poly A(+) RNA from developing bean seeds and isolated 2 nearly full-length cDNA clones (813 and 826 bp long). The cDNAs encode 2 distinct isoforms of bean TBP, PV1 and PV2, each with an open reading frame of 200 amino acid residues. The 2 cDNA sequences share an 85.8% overall nucleotide sequence identity, with the coding region showing a higher degree of identity (94.4%) than the 5′- and 3′-untranslated regions (69%). The deduced amino acid sequence of the bean TBP isoforms differ in only 3 amino acid residues at positions 5, 9, and 16, all located in the amino-terminal region. The carboxyl-terminal domain of 180 amino acid residues shows a high degree (>82%) of evolutionary sequence conservation with the TBP sequences from other eukaryotic species. This domain possesses the 3 highly conserved structural motifs, namely the 2 direct repeat sequences, a central basic region rich in basic amino acid residues, and a region similar to the sigma factor of prokaryote. On the basis of this and other findings, we suggest that higher plants in general may have at least 2 copies of TBP gene, presumably resulting from the global duplication of the genome. Accession numbers AF015784 and AF015785 at the GenBank.     

Genes involved in aniline degradation by Delftia acidovorans strain 7N and its distribution in the natural environment

Aniline-degraders were isolated from activated sludge and environmental samples and classified into eight phylogenetic groups. Seven groups were classified into Gram-negative bacteria, such as Acidovorax sp., Acinetobacter sp., Delftia sp., Comamonas sp., and Pseudomonas sp., suggesting the possible dominance of Gram-negative aniline-degraders in the environment. Aniline degradative genes were cloned from D. acidovorans strain 7N, and the nucleotide sequence of the 8,039-bp fragment containing eight open reading frames was determined. Their deduced amino acid sequences showed homologies to glutamine synthetase (GS)-like protein, glutamine amidotransferase (GA)-like protein, large and small subunits of aniline dioxygenase, reductase, LysR-type regulator, small ferredoxin-like protein, and catechol 2,3-dioxygenase, suggesting a high similarity of this gene cluster to those in P. putida strain UCC22 and Acinetobacter sp. strain YAA. Polymerase chain reaction (PCR) and sequencing analyses of GS-like protein gene segments of other Gram-negative bacteria suggested that Gram-negative bacteria have aniline degradative gene that can be divided into two distinctive groups.     

Homology cloning of protein kinase and phosphoprotein phosphatase sequences of Dictyostelium discoideum.

Reversible protein phosphorylation appears to be important at several stages in the signal transduction pathways in Dictyostelium discoideum. To elucidate its role, we have isolated sequences encoding putative protein kinases and phosphoprotein phosphatases by homology cloning using polymerase chain reactions (PCRs). Oligonucleotide primers were synthesized for use as forward and reverse primers with their nucleotide sequences deduced from the amino acid sequences of conserved domains of several protein kinases and phosphoprotein phosphatases. The fragments amplified by PCR were cloned, sequenced, and shown to encode parts of five different protein kinases and two phosphoprotein phosphatases. Several features such as the deduced amino acid sequence homology, location of invariant amino acids, GC content, and the codon usage confirmed that one set of clones encode parts of different protein kinases of Dictyostelium. Two clones derived from phosphoprotein phosphatase primers encode fragments of type 1 and type 2A phosphoprotein phosphatases. Amplified fragments were used to screen a lambda gt11 bank, and several cDNA clones for protein kinases were isolated. Some of these show differential expression during development or in response to exogenous cAMP.     

Cloning of a novel pilin-like gene from Bordetella pertussis: homology to the fim2 gene

A search for pilin genes in a Bordetella pertussis (Bp) genomic library has led to the identification of several clones which hybridize to synthetic oligonucleotides with sequences derived from amino acid sequences of Bp fimbrial subunits. One of these clones (corresponding to a gene we have named fimX) contains an open reading frame encoding a protein with a molecular weight of about 20 kD and a sequence similar but not identical to the fimbrial subunit fim2 and to other fimbrial protein sequences. In this communication we present the cloning and nucleotide sequence of the fimX gene and its homology to the fim2 gene. A genomic analysis on the positional relationship between the two genes is also presented.     

Combined Molecular and Conventional Analyses of Nitrifying Bacterium Diversity in Activated Sludge: Nitrosococcus mobilis and Nitrospira-Like Bacteria as Dominant Populations

The ammonia-oxidizing and nitrite-oxidizing bacterial populations occurring in the nitrifying activated sludge of an industrial wastewater treatment plant receiving sewage with high ammonia concentrations were studied by use of a polyphasic approach. In situ hybridization with a set of hierarchical 16S rRNA-targeted probes for ammonia-oxidizing bacteria revealed the dominance of Nitrosococcus mobilis-like bacteria. The phylogenetic affiliation suggested by fluorescent in situ hybridization (FISH) was confirmed by isolation of N. mobilis as the numerically dominant ammonia oxidizer and subsequent comparative 16S rRNA gene (rDNA) sequence and DNA-DNA hybridization analyses. For molecular fine-scale analysis of the ammonia-oxidizing population, a partial stretch of the gene encoding the active-site polypeptide of ammonia monooxygenase (amoA) was amplified from total DNA extracted from ammonia oxidizer isolates and from activated sludge. However, comparative sequence analysis of 13 amoA clone sequences from activated sludge demonstrated that these sequences were highly similar to each other and to the corresponding amoA gene fragments of Nitrosomonas europaea Nm50 and the N. mobilis isolate. The unexpected high sequence similarity between the amoA gene fragments of the N. mobilis isolate and N. europaea indicates a possible lateral gene transfer event. Although a Nitrobacter strain was isolated, members of the nitrite-oxidizing genus Nitrobacter were not detectable in the activated sludge by in situ hybridization. Therefore, we used the rRNA approach to investigate the abundance of other well-known nitrite-oxidizing bacterial genera. Three different methods were used for DNA extraction from the activated sludge. For each DNA preparation, almost full-length genes encoding small-subunit rRNA were separately amplified and used to generate three 16S rDNA libraries. By comparative sequence analysis, 2 of 60 randomly selected clones could be assigned to the nitrite-oxidizing bacteria of the genus Nitrospira. Based on these clone sequences, a specific 16S rRNA-targeted probe was developed. FISH of the activated sludge with this probe demonstrated that Nitrospira-like bacteria were present in significant numbers (9% of the total bacterial counts) and frequently occurred in coaggregated microcolonies with N. mobilis.