A study on the phylogeny and the ecology of ammonia-oxidizing bacteria using a new molecular marker based on the gene amoB

  1. Laia Calvó Perxas
Supervised by:
  1. L. Jesús García Gil Director

Defence university: Universitat de Girona

Year of defence: 2005

  1. Carlos Abella Ametller Chair
  2. Maria Dolors Balaguer Condom Secretary
  3. Emilio Ortega Casamayor Committee member
  4. Erik Lindgren Committee member
  5. David Rodríguez Lázaro Committee member

Type: Thesis

Teseo: 128957 DIALNET lock_openTDX editor


Human activities such as farming and industrialization have produced a significant increase in the number of ammonium-rich environments. The presence of nitrogenated compounds reduces water quality causing toxicity problems, deteriorating the environment and even affecting human health. Consequently, nitrification has recently become a widespread process involving the cycling of nitrogen in the biosphere, which is mainly due to microbial activities. Ammonia oxidizing bacteria (AOB) are an essential component of the global cycling of nitrogen, being responsible for the aerobic oxidation of ammonium to nitrite. Although the first ammonia oxidizers were isolated by the end of the XIX century, the slowness of their growth and the difficulties in culturing hindered achieving a full knowledge of this bacterial group until the 80s, when the first studies based on the gene 16S rDNA where performed. Nowadays, the databases contain huge numbers of entries of 16SrDNA sequences belonging to AOB. The aim of this work was to find, develop, and evaluate useful and reliable tools for the study of ammonia oxidizers in environmental samples. In this work we describe the use of Fluorescence In Situ Hybridization (FISH), based on the use of DNA probes specifically targeting the ammonia-oxidizers 16SrRNA molecule. AOB were detected and enumerated by using this technique. However, unknown sequences are hardly detectable by using this method, and therefore, new tools were needed. For this purpose we tried applying the sequence of the probe Nso1225 in a PCR reaction. The possibility of specifically amplifying a 16S rDNA gene fragment resulted in a new fingerprinting tool to assess the presence and diversity of ammonia-oxidizers in natural environments. Even so, some ßProteobacterial nonAOB sequences were also retrieved by using this technique. Moreover, one of the main disadvantages of using 16S rDNA as a molecular marker is the impossibility of simultaneously detecting both the ß and the ?Proteobacterial ammonia oxidizers. The gene amoA, which encodes for the subunit A of the enzyme ammonia monooxygenase, was then being extensively used as a marker for the detection of AOB in environmental samples. We describe the use of this marker for the identification of several ammonia oxidizing sequences in sludge samples from a sequencing batch reactor (SBR). Although useful, the use of amoA as a marker requires cloning, which is a tedious and time-consuming technique when dealing with large number of samples in microbial ecology studies. Besides, detection of nonAOB sequences has been reported by other authors when using amoA in a PCR-DGGE approach. Aiming at obtaining a fast and rigorous analytical tool allowing AOB detection and identification, we developed a new set of primers targeting the gene amoB, which encodes for the transmembrane domain of the enzyme ammonia monooxygenase. This gene has been shown to be a good molecular marker for AOB, since it can be used for easy detection and identification of ammonia oxidizers, providing high specificity, sensitivity and reliability regardless of phylogenetic affiliations. A real-time PCR assay for the detection and quantification of the ?proteobacterial genus Nitrosococcus based on the amoB gene sequence is also presented. This newly designed primer set allows a highly sensitive and specific enumeration of all known Nitrosococci. We finally performed a comparison and evaluation of the markers amoA, amoB and 16S rDNA, and built a polygenic based tree. As a result we conclude that amoB is a suitable molecular tool for detecting and identifying AOB in environmental samples, yielding consistent grouping when performing phylogenetic inferences. In turn, the whole sequence of the gene 16S rDNA is indicated for taxonomical and phylogenetic purposes when working with ammonia oxidizing isolates.