Patterns of community change among ammonia oxidizers in meadow soils upon long-term incubation at different temperatures

Abstract

The effect of temperature on the community structure of ammonia-oxidizing bacteria was investigated in three different meadow soils. Two of the soils (OMS and GMS) were acidic (pH 5.0 to 5.8) and from sites in Germany with low annual mean temperature (about 10 degrees C), while KMS soil was slightly alkaline (pH 7.9) and from a site in Israel with a high annual mean temperature (about 22 degrees C). The soils were fertilized and incubated for up to 20 weeks in a moist state and as a buffered (pH 7) slurry amended with urea at different incubation temperatures (4 to 37 degrees C). OMS soil was also incubated with less fertilizer than the other soils. The community structure of ammonia oxidizers was analyzed before and after incubation by denaturing gradient gel electrophoresis (DGGE) of the amoA gene, which codes for the alpha subunit of ammonia monooxygenase. All amoA gene sequences found belonged to the genus Nitrosospira. The analysis showed community change due to temperature both in moist soil and in the soil slurry. Two patterns of community change were observed. One pattern was a change between the different Nitrosospira clusters, which was observed in moist soil and slurry incubations of GMS and OMS. Nitrosospira AmoA cluster 1 was mainly detected below 30 degrees C, while Nitrosospira cluster 4 was predominant at 25 degrees C. Nitrosospira clusters 3a, 3b, and 9 dominated at 30 degrees C. The second pattern, observed in KMS, showed a community shift predominantly within a single Nitrosospira cluster. The sequences of the individual DGGE bands that exhibited different trends with temperature belonged almost exclusively to Nitrosospira cluster 3a. We conclude that ammonia oxidizer populations are influenced by temperature. In addition, we confirmed previous observations that N fertilizer also influences the community structure of ammonia oxidizers. Thus, Nitrosospira cluster 1 was absent in OMS soil treated with less fertilizer, while Nitrosospira cluster 9 was only found in the sample given less fertilizer.

FIG. 1.

Ammonium concentrations after incubation at different temperatures in high fertilizer (HF) treatments and low fertilizer (LF) treatments of OMS soil (Oppenrod, Germany). ▪, HF treatments after 6.5 weeks of incubation; •, LF treatments after 8 weeks of incubation; ▴, LF treatments after 16 weeks of incubation; and ▾, LF treatments after 20 weeks of incubation. Values are means ± standard error (n = 2).

FIG. 2.

Potential nitrification activity after 20 weeks of incubation at different temperatures. ▪, KMS soil; •, GMS soil; ▴, OMS soil high fertilizer (HF) treatment. Values are means ± standard error (n = 2).

FIG.3.

Fitch-Margoliash phylogenetic reconstruction (with global rearrangement and randomized input order) (three jumbles) based on partial AmoA sequences (150 amino acids) retrieved from KMS soil. Clones and bands obtained from this experiment are highlighted in bold. DNA was retrieved from field samples (FS) and the DGGE bands (band number) shown in Fig. 6. The designations of the clones and bands include the following information: t, incubation temperature; c, clone number; and b, band number. The scale bar indicates 10 changes per 100 nucleotide positions. The sequences of DGGE bands that are not mentioned in the tree due to >99% amino acid identity to another sequence: 1, KMSt20c6; 5, KMSt10c1; 6, KMSt37c18; and 10, KMSt37c1. Sequences from public databases are identified by their accession numbers. These sequences were published previously (1, 11, 15, 16, 19, 31, 32, 34, 36-38, 41, 43) or are unpublished sequences deposited in GenBank.

FIG. 4.

Fitch-Margoliash phylogenetic reconstruction (with global rearrangement and randomized input order) (three jumbles) based on partial AmoA sequences (150 amino acids) retrieved from GMS soil. Clones and bands obtained from this experiment are highlighted in bold. DNA was retrieved from field samples (FS) and the DGGE bands shown in Fig. 8. DGGE was conducted on DNA amplified from moist soil (GMS) and soil slurry (slGMS) incubations. Symbols: +, upper faint bands at 30°C of moist soil; *, upper faint bands at 25°C of moist soil; #, upperfaint bands at 25°C of slurry. The designations of the clones and bands include the following information: t, incubation temperature; c, clone number; and b, band number. The scale bar indicates 10 changes per 100 nucleotide positions. The sequences of DGGE bands that are not mentioned in the tree due to >99% amino acid identity to another sequence include: 1, GMSt25c41; 3, GMSt25c29; 4, GMSt25c10; 5, GMSt25c2; 8, GMSt30c4/slGMSt30b8; 9, slGMSt25c9; and 10, slGMSt25b10. Sequences from public databases are identified by their accession numbers. These sequences were published previously (1, 11, 15, 16, 19, 31, 32, 34, 36-38, 41, 43) or are unpublished sequences deposited in GenBank.

FIG.5.

Fitch-Margoliash phylogenetic reconstruction (with global rearrangement and randomized input order) (three jumbles) based on partial AmoA sequences (150 amino acids) retrieved from OMS soil. Clones and bands obtained from this experiment are highlighted in bold. DNA was retrieved from field sample (FS) and the DGGE bands shown in Fig. 9. DGGE was conducted on DNA amplified from moist soil (OMS) at both LF (OMSLF) and HF (OMSHF) treatments and from soil slurry (slOMS) incubations. The designations of the clones and bands include the following information: t, incubation temperature; c, clone number; and b, band number. The scale bar indicates 10 changes per 100 nucleotide positions. The sequences of DGGE bands that are not mentioned in the tree due to >99% amino acid identity to another sequence include: 1, slOMSt25b1; 2, OMSHFt25c16; 3, OMSHFt25b3; 4, OMSHFt30b4; 7, OMSHFt25b7; 8, OMSHFt25c7; 10, slOMSt20c35; 11, slOMSt25c1; 12, slOMSt15b12; 13, slOMSt25b13; 14, OMSLFt20c23; 15, OMSLFt15b15; 18, OMSLFt20b18; 21, OMSLFt30b21; 22, OMSLFt10c44; 23, OMSLFt20c14; 25, OMSLFt30b25; and 26, OMSLFt30b26. Sequences from public databases are identified by their accession numbers. These sequences were published previously (1, 11, 15, 16, 19, 31, 32, 34, 36-38, 41, 43) or are unpublished sequences deposited in GenBank.

FIG. 6.

DGGE analysis of amoA fragments retrieved from KMS soil after 16 and 20 weeks of incubation at different temperatures. Soil samples were from moist soil or slurry incubations. Band numbers are the same as in the corresponding AmoA tree in Fig. 3. Nt represents Nitrosospira tenuis as a reference bacterium.

FIG. 7.

Correspondence analysis comparing the differences in DGGE banding patterns with the program SYSTAT 9. Open circles represent samples of KMS soil which were incubated at different temperatures (4 to 37°C) in the moist soil state. The names of samples indicate the incubation temperature and period of incubation in weeks (w). Solid circles with a line represent bands with numbers in bold.

FIG. 8.

DGGE analysis of amoA fragments retrieved from GMS soil incubated for different times at different temperatures (4 to 30°C) as (a) moist soil and (b) soil slurry. Band numbers are the same as in the corresponding AmoA tree in Fig. 4. Zt, time zero of the experiment; Nt, Nitrosospira tenuis as a reference bacterium.

FIG. 9.

DGGE analysis of amoA fragments retrieved from OMS soil incubated for different times at different temperatures (4 to 30°C). Soil samples were from slurry and moist soil incubations at (a) high fertilizer (HF) treatment and (b) low fertilizer (LF) treatment. As a comparison, three samples from slurry incubations after 12.5 weeks (15°C) and 19.5 weeks (25°C) were loaded on the gel with LF-treated moist soil. Band numbers are the same as in the corresponding AmoA tree in Fig. 5. Nt, Nitrosospira tenuis as a reference bacterium.

FIG. 10.

Correspondence analysis comparing the differences in DGGE banding patterns with the program SYSTAT 9. Open circles represent samples of OMS soil which were incubated at different temperatures (4 to 30°C) and/or in different ammonium treatments. The names of samples indicate the temperature, slurry (S), low fertilizer treatment (LF), high fertilizer treatment (HF), and period of incubation in weeks (w). Solid circles with a line represent DGGE bands with numbers in bold. Bands 15, 17, and 21 are not shown because they are identical in amino acid sequence to bands 3, 7, and 9, respectively, but their contribution to the samples was considered in the calculations.