Richard Hastings

Amplification of 16S ribosomal RNA genes of autotrophic ammonia-oxidizing bacteria demonstrates the ubiquity of nitrosospiras in the environment

Oligonucleotide sequences selected from the 16S rRNA genes of various species of ammonia-oxidizing bacteria were evaluated as specific PCR amplification primers and probes. The specificities of primer pairs for eubacterial, Nitrosospira and Nitrosomonas rRNA genes were established with sequence databases, and the primer pairs were used to amplify DNA from laboratory cultures and environmental samples. Eubacterial rRNA genes amplified from samples of soil and activated sludge hybridized with an oligonucleotide probe specific for Nitrosospira spp., but not with a Nitrosomonas-specific probe. Lakewater and sediment samples were analysed using a nested PCR technique in which eubacterial rRNA genes were subjected to a secondary amplification with Nitrosomonas or Nitrosospira specific primers. Again, the presence of Nitrosospira DNA, but not Nitrosomonas DNA, was detected and this was confirmed by hybridization of the amplified DNA with an internal oligonucleotide probe. Enrichments of lakewater and sediment samples, incubated for two weeks in the presence of ammonium, produced nitrite and were found to contain DNA from both Nitrosospira and Nitrosomonas as determined by nested PCR amplification and probing of 16S rRNA genes. This demonstrates that Nitrosospira spp. are widespread in the environment. The implications of the detection of Nitrosomonas DNA only after enrichment culture are discussed.

Analysis of ammonia-oxidizing bacteria populations in acid forest soil during conditions of moisture limitation

R.C. HASTINGS, C. BUTLER, I. SINGLETON, J.R. SAUNDERS and A.J. McCARTHY.2000.Ammonia‐oxidizer numbers decreased under conditions of moisture limitation in litter, fermentation and humus layers of forest soil in the field, but the extent of regrowth after rehydration varied between layers. Nitrosospira 16S rRNA genes were amplified from all layers, regardless of moisture content or soil pH which varied between 4·1 and 5·2. Nitrosomonas spp. were detected less often, but appeared to exhibit more rapid recovery than the Nitrosospira spp. when drought conditions were relieved by rainfall.

Effects of soil improvement treatments on bacterial community structure and soil processes in an upland grassland soil

Abstract Temporal temperature gradient electrophoresis (TTGE) analysis of 16S rRNA gene fragments amplified with primers selective for eubacteria and beta-proteobacterial ammonia-oxidising bacteria (AOB) was used to analyse changes in bacterial and AOB community profiles of an upland pasture following soil improvement treatments (addition of sewage sludge and/or lime). Community structure was compared with changes in activity assessed by laboratory measurements of basal respiration and ammonia oxidation potentials, and with measurements of treatment- and time-related changes in soil characteristics. The predominant bacterial populations had a high degree of similarity under all treatment regimens, which was most pronounced early in the growing season. Most of the differences that occurred between soil samples with time could be accounted for by spatial and temporal variation; however, analysis of variance and cluster analysis of similarities between 16S rDNA TTGE profiles indicated that soil improvement treatments exerted some effect on community structure. Lime application had the greatest influence. The impact of soil improvement treatments on autotrophic ammonia oxidation was significant and sustained, especially in soils which had received sewage sludge and lime treatments in combination. However, despite obvious changes in soil characteristics, e.g. pH and soil nitrogen, increasing heterogeneity in the AOB community structure over time obscured the treatment effects observed at the beginning of the experiment. Nevertheless, time series analysis of AOB TTGE profiles indicated that the AOB community in improved soils was more dynamic than in control soils where populations were found to be relatively stable. These observations suggest that the AOB populations exhibited a degree of functional redundancy.

Effects of swine manure fertilization on autotrophic ammonia oxidizing bacteria in soil

Abstract Molecular and biochemical investigation methods have been exploited to evaluate the effects of pig slurry, used for three years as a fertilizer in intensive agriculture, on the presence and activity of chemolithotrophic ammonia oxidizing bacteria, the biocatalysts of the first step in the nitrification process. The evaluation was carried out, on bulk soil, comparing data from the first and the third year of fertilization. Oligonucleotide sequences selected from the 16S rRNA genes of autotrophic ammonia oxidizers have been used as specific PCR amplification primers and probes. The potential nitrification activity (PNA) has been used to determine the effects of swine manure fertilization on the ammonia oxidizing activity. Members of the genera Nitrosomonas and Nitrosospira were always detectable, but the genus Nitrosospira was more represented than Nitrosomonas . The differences between the hybridization signals were correlated with the doses of pig slurry and the number of applications. The mineral (urea) and organic fertilizations showed similar effects on the ammonia oxidisers investigated by molecular and biochemical methods. It was found that additions of swine manure to soil plots increased both potential ammonia-oxidise activity and ammonia oxidiser sequences. Stimulation of the soil ammonia-oxidise activity was due mainly to its effects on the indigenous bacterial population.

Campylobacter genotypes from poultry transportation crates indicate a source of contamination and transmission

Aims:  Crates used to transport live poultry can be contaminated with Campylobacter, despite periodic sanitization, and are potential vectors for transmission between flocks. We investigated the microbial contamination of standard and silver ion containing crates in normal use and the genetic structure of associated Campylobacter populations.