A 16s rRNA-based molecular ecological study was performed to search for dominant bacterial sequences in Drentse A grassland soils (The Netherlands). In the first step, a library of 165 clones was generated from PCR-amplified 165 rDNA. By sequence comparison, clone DA079 and two other identical clones could be affiliated to a group of recently described uncultured Actinobacteria. This group contained 165 rDNA clone sequences obtained from different environments across the world. To determine whether such uncultured organisms were part of the physiologically active population in the soil, ribosomes were isolated from the environment and 16s rRNA was partially amplified via RT-PCR using conserved primers for members of the domain Bacteria. Subsequent sequence-specif ic separation by temperature-gradient gel electrophoresis (TGGE) generated fingerprints of the amplicons. Such community fingerprints were compared with the TGGE pattern of PCRamplified rDNA of clone DA079 which was generated with the same set of primers. One of the dominant fingerprint bands matched with the band obtained from the actinobacterial clone. Southern blot hybridization with a probe made from clone DA079 confirmed sequence identity of clone and fingerprint band. This is the first report that a member of the novel actinobacterial group may play a physiologically active role in a native microbial community.

The main bacteria in peaty, acid grassland soils in the Netherlands were investigated by ribosome isolation, temperature gradient gel electrophoresis, hybridization, cloning, and sequencing. Instead of using only 16S rDNA to determine the sequences present, we focused on rRNA to classify and quantify the most active bacteria. After direct ribosome isolation from soil, a partial amplicon of bacterial 16S rRNA was generated by reverse transcription-PCR. The sequence-specific separation by temperature gradient gel electrophoresis yielded soil-specific fingerprints, which were compared to signals from a clone library of genes coding for 16S rRNA. Cloned 16S rDNA sequences matching with intense bands in the fingerprint were sequenced. The relationships of the sequences to those of cultured organisms of known phylogeny were determined. Most of the amplicons originated from organisms closely related to Bacillus species. Such sequences were also detected by direct dot blot hybridization on soil rRNA: a probe specific for Firmicutes with low G+C content counted for about 50% of all bacterial rRNA. The bacterial activity in Drentse A grassland soil could be estimated by direct dot blot hybridization and sequencing of clones; it was found that about 65% of all the bacterial ribosomes originated from Firmicutes. The most active bacteria apparently were Bacillus species, from which about half of the sequences derived. Other sequences similar to those of gram-positive bacteria were only remotely related to known Firmicutes with a high G+C content. Other sequences were related to Proteobacteria, mainly the alpha subclass.

The composition of predominant soil bacteria during grassland succession was investigated in the Dutch Drentse A area. Five meadows, taken out of agricultural production at different time points, and one currently fertilized plot represented different stages of grassland succession. Since fertilization and agricultural production were stopped, the six plots showed a constant decline in the levels of nutrients and vegetation changes. The activity of the predominant bacteria was monitored by direct ribosome isolation from soil and temperature gradient gel electrophoresis of reverse transcription (RT)-PCR products generated from bacterial 16S rRNA. The amounts of 16S rRNA of 20 predominant ribosome types per gram of soil were monitored via multiple competitive RT-PCR in six plots at different succession stages. These ribosome types mainly represented Bacillus and members of the Acidobacterium cluster and the ␣ subclass of the class Proteobacteria. The 20 16S rRNA molecules monitored represented approximately half of all bacterial soil rRNA which was estimated by dot blot hybridizations of soil rRNA with the Bacteria probe EUB338. The grasslands showed highly reproducible and specific shifts of bacterial ribosome type composition. The total bacterial ribosome level increased during the first years after agricultural production and fertilization stopped. This correlated with the collapse of the dominant Lolium perenne population and an increased rate of mineralization of organic matter. The results indicate that there is a true correlation between the total activity of the bacterial community in soil and the amount of bacterial ribosomes.The agricultural overproduction of the last decades in Western Europe resulted in the release of more and more land from agricultural management, and many meadows providing hay for cattle feeding were left as unfertilized grassland. Further management of these areas was aimed at restoring the former species-rich vegetation by nonextensive hay making at moderate frequency. A well-studied model system to monitor this process is the Drentse A grassland research area. In the history of the Drentse A grasslands three different periods can be distinguished based on changing hay-making and fertilization practices (21). Until the 1930s, these grasslands showed a species-rich vegetation and were cut once or twice a year for hay production without application of chemical fertilizers. Then the agricultural use was intensified by increasing the cutting frequency and raising the hay production by applying artificial mineral fertilizers, resulting in domination by high-yield grass species and an overall decrease in species richness. Since the late 1960s part of the land has been released from agricultural production to restore the former species-rich vegetation by taking off hay only once a year without any fertilizer application. Today, different stages of this succession can be observed, since over the years more and more plots were added to the restoration management process. At the time of sam...

Dechloromonas hortensis sp. nov. and strain ASK-1, two novel (per)chlorate-reducing bacteria, and taxonomic description of strain GR-1 Recent studies on the occurrence of (per)chlorate-reducing bacteria have resulted in the characterization of strains capable of dissimilatory (per)chlorate reduction. Phylogenetic analysis has shown that these bacteria are members of the Proteobacteria. Strains have been isolated from polluted and pristine sites, but only strains from polluted sites have been characterized in detail and deposited in culture collections. Herein we describe the isolation and characterization of perchlorate-reducing bacterium strain MA-1 T and chlorate-reducing bacterium strain ASK-1, respectively isolated from a pristine and a chlorate-polluted site. Both isolates are members of the Proteobacteria. The 16S rRNA gene sequence similarity of MA-1 T to Dechloromonas agitata DSM 13637 T is 97?6 %, but the relatedness in DNA-DNA reassociation is only 37 %. Therefore, we propose to classify strain MA-1 T (=DSM 15637 T =ATCC BAA-776 T ) as the type strain of a novel species, Dechloromonas hortensis sp. nov. Strain ASK-1 and a previously described strain GR-1 show 100 and 99 % 16S rRNA gene sequence similarity to Pseudomonas chloritidismutans DSM 13592 T and Dechlorosoma suillum DSM 13638 T , respectively. DNA-DNA hybridization studies indicated that strains ASK-1 and GR-1 are related at the species level to P. chloritidismutans DSM 13592 T (79 %) and Dechlorosoma suillum DSM 13638 T (85 %), respectively. As suggested previously, Dechlorosoma suillum appears to be a later heterotypic synonym of Azospira oryzae. Although strain ASK-1 is identified as P. chloritidismutans, its morphology and growth requirements are different from those of the type strain.The occurrence of perchlorate and chlorate in natural environments is due mainly to human activities. Chlorate is used as a herbicide, as a catalyst in matches and for onsite production of chlorine dioxide (ClO 2 ), a bleaching agent employed in the paper and pulp industries. Perchlorate is used as rocket propellant in the defence and aerospace industries. In the United States, discharge of perchlorate-containing waste streams has been identified as the major source of perchlorate contamination in drinking water supplies (Renner, 1998; US Environmental Protection Agency, 2000). Chlorate and perchlorate are highly soluble and chemically stable under environmental conditions (Urbansky, 2002). Remediation strategies by chemical reduction or by adsorption to activated carbon are either too slow or incomplete (Urbansky, 1998). Recent research has shown that bioremediation may be the most economically feasible, fastest and easiest means of treating (per)chlorate-contaminated sites and water sources (Coates & Achenbach, 2004). (Per)chlorate-reducing bacteria are able to perform aThe GenBank/EMBL/DDBJ accession numbers of the 16S rRNA gene sequences of strains ASK-1, MA-1 and GR-1 are AY277620, AY277621 and AY277622.Photographs of colonies and cell extracts of P. chl...

The chlorite dismutase (Cld) of Pseudomonas chloritidismutans was purified from the periplasmic fraction in one step by hydroxyapatite chromatography. The enzyme has a molecular mass of 110 kDa and consists of four 31-kDa subunits. Enzyme catalysis followed Michaelis-Menten kinetics, with Vmax and K(m) values of 443 U mg(-1) and 84 microM, respectively. A pyridine-NaOH-dithionite-reduced Cld revealed a Soret peak at 418 nm, indicative for protoheme IX. The spectral data indicate the presence of 1.5 mol protoheme IX mol(-1) tetrameric enzyme while metal analysis revealed 2.2 mol iron mol(-1) tetrameric enzyme. High concentrations of chlorite resulted in the disappearance of the Soret peak, which coincided with loss in activity. Electron paramagnetic resonance analyses showed an axial high-spin ferric iron signal. Cld was inhibited by cyanide, azide, but not by hydroxylamine or 3-amino-1,2,3-triazole. Remarkably, the activity was drastically enhanced by kosmotropic salts, and chaotropic salts decreased the activity, in accordance with the Hofmeister series. Chlorite conversion in the presence of 18O-labeled water did not result in the formation of oxygen with a mass of 34 (16O-18O) or a mass of 36 ((18)O-(18)O), indicating that water is not a substrate in the reaction and that both oxygen atoms originate from chlorite.