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. 1999 Jan;65(1):95-101.
doi: 10.1128/AEM.65.1.95-101.1999.

Effect of toxic metals on indigenous soil beta-subgroup proteobacterium ammonia oxidizer community structure and protection against toxicity by inoculated metal-resistant bacteria

J R Stephen  1 , Y J Chang, S J Macnaughton, G A Kowalchuk, K T Leung, C A Flemming, D C White
Affiliations

Effect of toxic metals on indigenous soil beta-subgroup proteobacterium ammonia oxidizer community structure and protection against toxicity by inoculated metal-resistant bacteria

J R Stephen et al. Appl Environ Microbiol. 1999 Jan.

Abstract

Contamination of soils with toxic metals is a major problem on military, industrial, and mining sites worldwide. Of particular interest to the field of bioremediation is the selection of biological markers for the end point of remediation. In this microcosm study, we focus on the effect of addition of a mixture of toxic metals (cadmium, cobalt, cesium, and strontium as chlorides) to soil on the population structure and size of the ammonia oxidizers that are members of the beta subgroup of the Proteobacteria (beta-subgroup ammonia oxidizers). In a parallel experiment, the soils were also treated by the addition of five strains of metal-resistant heterotrophic bacteria. Effects on nitrogen cycling were measured by monitoring the NH3 and NH4+ levels in soil samples. The gene encoding the alpha-subunit of ammonia monooxygenase (amoA) was selected as a functional molecular marker for the beta-subgroup ammonia oxidizing bacteria. Community structure comparisons were performed with clone libraries of PCR-amplified fragments of amoA recovered from contaminated and control microcosms for 8 weeks. Analysis was performed by restriction digestion and sequence comparison. The abundance of ammonia oxidizers in these microcosms was also monitored by competitive PCR. All amoA gene fragments recovered grouped with sequences derived from cultured Nitrosospira. These comprised four novel sequence clusters and a single unique clone. Specific changes in the community structure of beta-subgroup ammonia oxidizers were associated with the addition of metals. These changes were not seen in the presence of the inoculated metal-resistant bacteria. Neither treatment significantly altered the total number of beta-subgroup ammonia-oxidizing cells per gram of soil compared to untreated controls. Following an initial decrease in concentration, ammonia began to accumulate in metal-treated soils toward the end of the experiment.

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Figures

FIG. 1
FIG. 1
Ammonia and ammonium levels in soil microcosms. Ammonia and ammonium levels were determined as described previously (36). Week 0 values are given for the combined analysis of all microcosms (n = 12). All others are given for triplicate microcosms. NH3 and NH4+ levels fell rapidly in uncontaminated soils and stabilized by week 4.NH3 and NH4+ levels fell more slowly in metal-contaminated microcosms and then accumulated between weeks 4 and 8.
FIG. 2
FIG. 2
Restriction digestion analysis of amoA clones with MspI. Cloned amoA gene fragments were amplified from the cloning vector by using primers directed at the T7 and M13 reverse RNA polymerase binding sites, producing a fragment with approximately 70 bp of vector sequence on each end. The vector sequence contained no MspI recognition sites. Products were digested with a twofold excess of MspI for 1 h, analyzed by electrophoresis on a 2% agarose gel with TAE buffer, and visualized by ethidium bromide fluorescence. Lanes: 1, molecular size marker (100-bp ladder; Boehringer); 2, 9, and 19 pattern 1 amoA clones; 10, 17, and 18, pattern 2 amoA clones; 3 to 8, 11 to 16, and 20, pattern 3 amoA clones.
FIG. 3
FIG. 3
Neighbor-joining tree with Fitch-Margoliash correction of amoA sequences recovered from soil microcosms. The tree is derived from a compilation of available amoA sequences spanning the 449 nucleotide bases available for all sequences used. Sequences prefixed with NAB were generated during this study (accession no. AF056049 to AF056069). Clones were selected from libraries on the basis of MspI restriction patterns to provide a survey of sequence diversity in the target amoA sequences. Sequences which are identical over the fragment analyzed are presented on the same branch and separated by commas. Reference sequences: sequences SCHOH-SEE and PLUSS-SEE and sequences prefixed with RR and SP (environmental clones) (33); Nitrosospira briensis C57, Nitrosovibrio tenuis NV1, and Nitrosomonas europaea C-91 (pure cultures) (33); Nitrosospira sp. strain AHB1 (32); Nitrosomonas eutropha copies A1 and A2: (accession no. U51630 and U72670) (25); Nitrosospira multiformis copies A1, A2 and A3 (accession no. U91603, U15733, and U89833 respectively) (25); Nitrosospira briensis C128 (accession no. U76553) (25); Nitrosovibrio tenuis NV12 (accession no. U76552) (25); Nitrosospira sp. strain 39-19 copies A2 and A3 (accession no. AF016002 and AF006692, respectively) (25); Nitrosomonas europaea (23).
FIG. 4
FIG. 4
Proportions of sequence types in clone libraries. The library population structures of all week 8 samples were significantly different from the starting population (chi-square probabilities of <0.05, <0.0002, <0.05, and <0.05, respectively). The only significantly different population within the week 8 clone libraries was between the metal-treated soil without added inoculum and all other week 8 libraries (P = <0.003). Error bars represent standard deviations between libraries generated from duplicate microcosms. Chi-square values were calculated on the combined data.
FIG. 5
FIG. 5
(a) Quantification of amoA target sequences by competitive PCR. Results shown are from three soil microcosms at week 0 with added metals and inoculum. Lane 1 is a molecular size marker (1 kb-plus; Boehringer). Numbers indicate the number of linear amoA deletion fragments added to the reaction mixture. This generated the lower band visible in each lane. The relative abundances of amoA and amoA deletion fragments were assayed by ethidium bromide fluorescence and quantification with Alpha Imager software (Alpha Innotech). (b) Changes in amoA target numbers per gram of metal-contaminated soil microcosms. Values are averages of three reactions on DNA samples extracted from triplicate microcosms. Error bars indicate standard deviation. The prefix shows the time point: 0 indicates samples taken at week zero; 4 indicates samples taken at week 4. The suffix shows the treatment designation: 1, no inoculum, no metals; 2, inoculum added, no metals; 3, no inoculum, metals added; 4, inoculum added, metals added. Inoculation with metal-resistant bacteria did not significantly affect the total number of amoA target sequences per gram of soil detected by competitive PCR after 4 weeks.
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