Alexander Neef

Analysis of bacterial community structure in bulk soil by in situ hybridization

In situ hybridization with rRNA-targeted, fluorescent (Cy3-labeled) oligonucleotide probes was used to analyze bacterial community structure in ethanol- or paraformaldehyde-fixed bulk soil after homogenization of soil samples in 0.1% pyrophosphate by mild ultrasonic treatment. In ethanol-fixed samples 37 ± 7%, and in paraformaldehyde 41 ± 8% of the 4′, 6-diamidino-2-phenylindole(DAPI)-stained cells were detected with the bacterial probe Eub338. The yield could not be increased by enzymatic and/or chemical pretreatments known to enhance the permeability of bacterial cells for probes. However, during storage in ethanol for 7 months, the detectability of bacteria increased in both ethanol- and paraformaldehyde-fixed samples to up to 47 ± 8% due to an increase in the detection yield of members of the α-subdivision of Proteobacteria from 2 ± 1% to 10 ± 3%. Approximately half of the bacteria detected by probe Eub338 could be affiliated to major phylogenetic groups such as the α-, β-, γ-, and δ-subdivisions of Proteobacteria, gram-positive bacteria with a high G+C DNA content, bacteria of the Cytophaga-Flavobacterium cluster of the CFB phylum, and the planctomycetes. The analysis revealed that bacteria of the α- and δ-subdivision of Proteobacteria and the planctomycetes were predominant. Here, members of the α-subdivision of Proteobacteria accounted for approximately 10 ± 3% of DAPI-stained cells, which corresponded to 44 ± 16 × 108 cells (g soil, dry wt.)–1, while members of the δ-subdivision of Proteobacteria made up 4 ± 2% of DAPI-stained cells [17 ± 9 × 108 cells (g soil, dry wt.)–1]. A large population of bacteria in bulk soil was represented by the planctomycetes, which accounted for 7 ± 3% of DAPI-stained cells [32 ± 12 × 108 cells (g soil, dry wt.)–1]. The detection of planctomycetes in soil confirms previous reports on the occurrence of planctomycetes in soil and indicates a yet unknown ecological significance of this group, which to date has never been isolated from terrestrial environments.

Differential detection of key enzymes of polyaromatic-hydrocarbon-degrading bacteria using PCR and gene probes

Bacteria with ability to degrade polyaromatic hydrocarbons (PAHs), isolated from wastewater and soil samples, were investigated for their taxonomic, physiological and genetic diversity. Eighteen isolates able to metabolize naphthalene or phenanthrene as sole carbon source were taxonomically affiliated to different subclasses of the Proteobacteria (Sphingomonas spp., Acidovorax spp., Comamonas spp. and Pseudomonas spp.) and to phyla of Gram-positive bacteria with low and high DNA G + C content (Paenibacillus sp. and Rhodococcus spp., respectively). Representatives of the genera Pseudomonas and Sphingomonas formed a remarkably high fraction of these isolates; 9 out of 18 strains belonged to these groups. Tests for enzyme activities showed that the majority of the isolates growing with PAHs as sole sources of carbon and energy had an active catechol 2,3-dioxygenase (C230). C230 specific activities were very diverse, ranging from 0.1 to 650 mU (mg protein)-1. Pseudomonas and Sphingomonas strains showed considerably higher activities than the other isolates. All PAH degraders were examined for the presence of an initial PAH dioxygenase and C230, which catalyse key steps of PAH degradation, by PCR amplification of gene fragments and subsequent hybridization. PCR primers and internal oligonucleotide probes were developed for the specific detection of the genes of Pseudomonas and Sphingomonas strains.

Detection of sphingomonads and in situ identification in activated sludge using 16S rRNA-targeted oligonucleotide probes

The increasing significance of members of the genus Sphingomonas in biotechnological applications has led to an increased interest in the diversity, abundance and ecophysiological potential of this group of Gram-negative bacteria. This general focus provides a challenge to improve means for identification of sphingomonads; eg molecular genetic methods for rapid and specific detection could facilitate screening of new isolates. Here, fluorescently labeled oligonucleotide probes targeted against 16S rRNA were used to typify strains previously assigned to the genus. All 46 sphingomonads tested including type strains of 21 Sphingomonasspecies could be detected with a probe originally designed for the genus and all but one with a probe designed for the alpha-4 subgroup of the Proteobacteria. The two probes are suitable for direct detection of sphingomonads in pure and mixed cultures as well as in environmental samples of unknown composition. The probes were used to identify sphingomonads in situ in activated sludge samples. Sphingomonads were rather abundant accounting for about 5–10% of the total cells in municipal sludges. Distinct patterns in aggregation of the cells suggest that these organisms could be involved in the formation process of sludge flocs.

rRNA based identification and detection systems for rhizobia and other bacteria

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.

Denitrification in a methanol-fed fixed-bed reactor. Part 2: Composition and ecology of the bacterial community in the biofilms

Abstract In a methanol-fed post-denitrification biofilm system, the density and structure of the denitrifying bacterial community were investigated. Denitrification was operated in a sand filter succeeding a municipal two-stage wastewater treatment with full nitrification. The quantitative and qualitative analysis of the community structure revealed distinct taxonomic groups to be involved in denitrification. Autecological data on these bacteria indicate potential roles in the filter system which help to control operation. Members of the genera Hydrogenophaga and Comamonas of the β-subclass of Proteobacteria seem to proliferate at both aerobic and anoxic conditions with non-C1-carbon sources being available, i.e. in the upper layers of the filter bed with biomass being washed in from the secondary clarifiers. It is likely that a high number of the β-subclass bacteria is washed in and does not belong to the autochthonous flora of the denitrifying biofilms. In contrast, members of the genus Paracoccus of the α-subclass are likely to constitute an important part of the autochthonous denitrifiers. They seem to be favoured at aerobic conditions with C1-sources being available, as is the case in the surface layers of the biofilms. Moreover, methylotrophic members of the genus Hyphomicrobium are supposed to belong to the autochthonous flora. They are favoured by anoxic conditions and, therefore, are supposed to proliferate throughout the biofilms. The assumptions are verified by in-situ hybridization techniques. Both paracocci and hyphomicrobia are sensitive to pH drops in the filter bed, e.g. by acid production in thick biofilms, which should strictly be avoided. For details on physico-chemical and biomass parameters of the filter cells in situ see Part 1.

Detection of microbial cells in aerosols using nucleic acid probes

Summary Non-radioactive hybridization methods were evaluated for the identification of microorganisms in mixed bioaerosols. A cultivation-dependent method, colony hybridization, was compared to a direct, cultivation-independent approach, whole cell hybridization with fluorescently labeled oligonucleotides. After sampling of the aerosols by filtration, special processing of filters (cells) preceded hybridization with fluorescently, digoxigenin- or enzyme-labeled oligonuculeotide probes. Group, genus, or species affiliation of collected cells was analyzed with rRNA-targeted probes. Using nucleic acid probes directed against the multiple cloning site, plasmid bearing Escherichia coli colonies could be differentiated from wild-type colonies. The microbial composition of aerosols ranging from less than one to greater than 10 9 cells/m 3 air could be analyzed with appropriate hybridization formats: whole cell hybridization was only applicable to dense aerosols, colony hybridization yielded best results with lower concentrations. After a short incubation period (several hours), a combination of both formats could be used to rapidly determine the fraction of culturable cells within a bioaerosol. When applying these techniques for the monitoring of aerosols generated by standard microbiological laboratory procedures, low concentrations of airborne Escherichia coli cells (1–450 m −3 ) could be detected. Compared to conventional air monitoring techniques, hybridization with nucleic acid probes should allow more rapid and reliable detection of airborne microorganisms including genetic engineered microorganisms.

Novosphingobium hassiacum sp. nov., a New Species Isolated from an Aerated Sewage Pond

The taxonomy of two strains W-51T and W-52 isolated from a wastewater treatment plant was investigated in a polyphasic approach. The yellow pigmented gram-negative organism contained a quinone system with mainly ubiquinone Q-10, and the polar lipid profile contained a sphingoglycolipid suggesting that both strains belonged to the the alpha-4 subclass of the Proteobacteria. The polar lipid profile consisted furthermore of phosphatidylethanolamine, diphosphatidylglycerol, and phosphatidylcholine and of minor amounts of phosphatidylglycerol and phosphatidylmonomethylethanolamine. Sequencing of the 16S rRNA gene supported the allocation into the genus Novosphingobium, together with the type strains of N. subterraneum, N. aromaticivorans, N. stygium, and N. capsulatum, showing similarities of 97.3%, 97.0%, 95.7% and 96.2%, respectively. This allocation was supported by the polyamine profile, which consisted mainly of spermidine. The analysis of the fatty acids revealed 2-OH 13:0, 2-OH 14:0 and 2-OH 15:0, with 2-OH 15:0 as predominant hydroxylated fatty acid. W-51T and W-52 were almost identical with respect to their phenotypic including the majority of the chemotaxonomic properties, identical in their 16S rRNA sequences, and showed 86% DNA-DNA similarity. Both strains were able to reduce nitrate and on the basis of further physiological features, a clear differentiation from all other Novosphingobium species was possible. The DNA-DNA similarities of W-51T to the type strains of N. subterraneum, N. aromaticivorans, and N. capsulatum were below 56%. For these reasons, it is proposed to create a new species with the name Novosphingobium hassiacum sp. nov.

Sphingopyxis witflariensis sp. nov., isolated from activated sludge.

Classification of strain W-50(T), which was isolated from a wastewater treatment plant, was investigated by a polyphasic approach. Cells of strain W-50(T) were Gram-negative, strictly aerobic, oxidase-positive and yellow-pigmented. Ubiquinone Q-10 was the main respiratory lipoquinone system and polar lipid fingerprints were characterized by the presence of a sphingoglycolipid, suggesting that strain W-50(T) belongs to the alpha-4 subclass of the Proteobacteria. Sequencing and comparative analyses of the 16S rRNA gene of strain W-50(T) supported its chemotaxonomic allocation as an alpha-4 proteobacterium. The most closely related established taxa were species of the genus Sphingopyxis, including Sphingopyxis macrogoltabida (97.3% similarity) and Sphingopyxis terrae (96-4% similarity), and Sphingomonas taejonensis (97.3%). These findings were supported by both the polyamine content, which consisted mainly of spermidine [12.9 micromol (g dry wt)(-1)], and the presence of 2-OH 14:0, 2-OH 15:0 and 2-OH 16:0 in the cellular fatty acid profile. DNA-DNA hybridization experiments resulted in similarity values of 31.9% between strain W-50(T) and Sphingopyxis macrogoltabida IFO 15033(T), 44.9% between strain W-50(T) and Sphingopyxis terrae IFO 15098(T) and 31.0% between strain W-50(T) and Sphingomonas taejonensis KCTC 2884(T). Based upon results obtained by detailed physiological/biochemical testing and previously published molecular evidence, strain W-50(T) was clearly distinguishable from all other Sphingopyxis species. For these reasons, the creation of a novel species, Sphingopyxis witflariensis sp. nov., is proposed; strain W-50(T) (= DSM 14551(T) = CIP 107174(T)) is the type strain.

Chelatobacter heintzii (Auling et al. 1993) is a later subjective synonym of Aminobacter aminovorans (Urakami et al. 1992).

Chelatobacter heintzii, which was described as a nitrilotriacetate-utilizing organism, was re-investigated in order to clarify its taxonomic position. On the basis of 16S rDNA sequence comparisons, it is obvious that this species clusters phylogenetically with species of the genus Aminobacter. The results of investigations of the fatty acid patterns, polar lipid profiles, polyamine patterns and quinone system supported this placement. The substrate-utilization profiles and fatty acid patterns of four strains (belonging to two different genomovars) revealed homogeneous results and showed high levels of similarity to Aminobacter aminovorans. DNA-DNA similarity studies confirmed that both genomovars of Chelatobacter heintzii belong to Aminobacter aminovorans. It could be shown that all species of this group are highly interrelated. On the basis of these data and previously published results, it is obvious that Chelatobacter heintzii is a later subjective synonym of Aminobacter aminovorans.

Fluorescence based rRNA sensor systems for detection of whole cells of Saccharomonospora spp. and Thermoactinomyces spp.

Airborne thermophilic actinomycetes (TPAs) are a growing hygienic challenge in different occupational situations e.g. large scale composting. This study describes first results of a new approach for highly specific and rapid detection of organisms of this group using fluorescently labelled oligonucleotide probes as sensors for whole cells. Three genus-specific 16S rRNA-targeted probes, two for Saccharomonospora spp. and one for Thermoactinomyces spp. were developed and evaluated in a fluorescence in situ hybridisation (FISH) format with agar-grown whole cells. For optimal sensitivity and specificity of FISH, conditions for cell wall permeabilisation and hybridisation stringency were evaluated independently for both genera. Performing specified pretreatment protocols, all three probes yielded strong fluorescence signals. However, the relative fraction of detectable cells or spores clearly depended on the single bacterial species. The probes can serve as cell sensors for direct detection of TPAs in natural samples.