Community structure of denitrifiers, bacteria, and archaea along redox gradients in Pacific Northwest marine sediments by terminal restriction fragment length polymorphism analysis of amplified nitrite reductase (nirS) and 16S rRNA genes.

Steep vertical gradients of oxidants (O(2) and NO(3)(-)) in Puget Sound and Washington continental margin sediments indicate that aerobic respiration and denitrification occur within the top few millimeters to centimeters. To systematically explore the underlying communities of denitrifiers, Bacteria, and Archaea along redox gradients at distant geographic locations, nitrite reductase (nirS) genes and bacterial and archaeal 16S rRNA genes (rDNAs) were PCR amplified and analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The suitablility of T-RFLP analysis for investigating communities of nirS-containing denitrifiers was established by the correspondence of dominant terminal restriction fragments (T-RFs) of nirS to computer-simulated T-RFs of nirS clones. These clones belonged to clusters II, III, and IV from the same cores and were analyzed in a previous study (G. Braker, J. Zhou, L. Wu, A. H. Devol, and J. M. Tiedje, Appl. Environ. Microbiol. 66:2096-2104, 2000). T-RFLP analysis of nirS and bacterial rDNA revealed a high level of functional and phylogenetic diversity, whereas the level of diversity of Archaea was lower. A comparison of T-RFLPs based on the presence or absence of T-RFs and correspondence analysis based on the frequencies and heights of T-RFs allowed us to group sediment samples according to the sampling location and thus clearly distinguish Puget Sound and the Washington margin populations. However, changes in community structure within sediment core sections during the transition from aerobic to anaerobic conditions were minor. Thus, within the top layers of marine sediments, redox gradients seem to result from the differential metabolic activities of populations of similar communities, probably through mixing by marine invertebrates rather than from the development of distinct communities.

Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples.

A system was developed for the detection of denitrifying bacteria by the amplification of specific nitrite reductase gene fragments with PCR. Primer sequences were found for the amplification of fragments from both nitrite reductase genes (nirK and nirS) after comparative sequence analysis. Whenever amplification was tried with these primers, the known nir type of denitrifying laboratory cultures could be confirmed. Likewise, the method allowed a determination of the nir type of five laboratory strains. The nirK gene could be amplified from Blastobacter denitrificans, Alcaligenes xylosoxidans, and Alcaligenes sp. (DSM 30128); the nirS gene was amplified from Alcaligenes eutrophus DSM 530 and from the denitrifying isolate IFAM 3698. For each of the two genes, at least one primer combination amplified successfully for all of the test strains. Specific amplification products were not obtained with nondenitrifying bacteria or with strains of the other nir type. The specificity of the amplified products was confirmed by subsequent sequencing. These results suggest the suitability of the method for the qualitative detection of denitrifying bacteria in environmental samples. This was shown by applying one generally amplifying primer combination for each nir gene developed in this study to total DNA preparations from aquatic habitats.

Monitoring the denitrifying Hyphomicrobium DNA/DNA hybridization group HG 27 in activated sludge and lake water using MPN cultivation and subsequent screening with the gene probe Hvu-1.

Gene probe Hvu-1 is specific for the Hyphomicrobium DNA/DNA-hybridization group HG 27. This group is one of the dominant populations of denitrifying hyphomicrobia in the activated sludge of the sewage treatment plant Plön. The wastewater treatment process of this treatment plant can be characterized as a combination of simultaneous and intermittent nitrification and denitrification. Using this specific probe Hvu-1 (combined with the most-probable-number method and colony hybridization) the abundance of this denitrifying Hyphomicrobium population in activated sludge and in the adjacent receiving Lake Kleiner Plöner See was investigated as a subfraction of total facultatively anaerobic hyphomicrobia. A 15-month monitoring of the activated sludge and of the lake water was conducted to determine temporal variations of the occurrence of this specific population. During the sampling period total facultatively anaerobic hyphomicrobia remained very constant over a year (9 x 10(4) to 6 x 10(5) ml-1). The population of the denitrifying Hyphomicrobium DNA/DNA-hybridization group HG 27 amounted to approximately 30% of the total facultatively anaerobic hyphomicrobia found in the activated sludge. Significant correlations to environmental background parameters or seasonal variations of this population were not found. Furthermore, this specific denitrifying Hyphomicrobium population has no importance for the lake ecosystem.

Development of a species-specific gene probe for Hyphomicrobium facilis with the inverse PCR.

A species-specific gene probe for Hyphomicrobium facilis was generated from a transposon Tn5-132 insertion mutant defective in methanol oxidation by the inverse PCR. With this probe, the abundance of H. facilis in a garden soil was determined as a subfraction of the total cultivable hyphomicrobia.