Enzyme production-based approach for determining the functions of microorganisms within a community

Abstract

The functions of specific microorganisms in a microbial community were investigated during the composting process. Cerasibacillus quisquiliarum strain BLx(T) and Bacillus thermoamylovorans strain BTa were isolated and characterized in our previous studies based on their dominance in the composting system. Strain BLx(T) degrades gelatin, while strain BTa degrades starch. We hypothesized that these strains play roles in gelatinase and amylase production, respectively. The relationship between changes in the abundance ratios of each strain and those of each enzyme activity during the composting process was examined to address this hypothesis. The increase in gelatinase activity in the compost followed a dramatic increase in the abundance ratio of strain BLx(T). Zymograph analysis demonstrated that the pattern of active gelatinase bands from strain BLx(T) was similar to that from the compost. Gelatinases from both BLx(T) and compost were partially purified and compared. Homologous N-terminal amino acid sequences were found in one of the gelatinases from strain BLx(T) and that of compost. These results indicate strain BLx(T) produces gelatinases during the composting process. Meanwhile, the increase in the abundance ratio of strain BTa was not concurrent with that of amylase activity in the compost. Moreover, the amylase activity pattern of strain BTa on the zymogram was different from that of the compost sample. These results imply that strain BTa may not produce amylases during the composting process. To our knowledge, this is the first report demonstrating that the function of a specific microorganism is directly linked to a function in the community, as determined by culture-independent and enzyme-level approaches.

FIG. 1.

Physical parameters of compost and degradation efficiency of kitchen refuse during the composting process. (A) Transition of temperature within compost. The arrow indicates day 36, when a portion of the compost was removed and new sawdust was added. (B) Transition of pH within the compost. (C) Degradation efficiency during the composting process. The average degradation efficiency for ∼7 days was calculated based on the dry weight (dw).

FIG. 2.

Microbial counts during the composting process. (A) Total counts were obtained from epifluorescence microscopic counts with BacLight (the sum of live cells and dead cells). The average of counts on three individual membranes was calculated. The error bars indicate standard errors. (B) Viability and culturability during the composting process. Viability (□) was expressed as a percentage of viable cell counts per total count obtained from microscopic counts. Culturability (×) was expressed as a percentage of CFU on TSA plates at 37°C per total count. The error bars indicate standard errors (n = 3 [viability] and n = 4 [culturability] CFU per total count).

FIG. 3.

Changes in the abundance ratio of strain BLx^T and gelatinase activity during the composting process. (A) The abundance ratio of strain BLx^T is represented as the DNA mass ratio (the DNA mass of strain BLx^T per DNA mass of the compost sample). The DNA mass of strain BLx^T was obtained from the quantitative real-time PCR-targeted 16S rDNA of strain BLx^T with a LightCycler system and genomic DNA from strain BLx^T. The error bars indicate standard errors (n ≥ 3). (B) Gelatinase activity was assayed with Azocoll as the substrate. The error bars indicate standard errors (n = 3). (C) Zymogram of gelatinase activities of compost samples and strain BLx^T culture supernatant with gelatin as the substrate. Each sample applied to the zymograph had the same number of gelatinase units based on the Azocoll assay.

FIG. 4.

Comparison of gelatinases present in strain BLx^T and the compost sample. (A) CBB-stained gel and zymograph gel. Samples were applied in equal amounts (10 μg of protein). Crude enzyme in culture supernatant from strain BLx^T (lanes 1 and 5) and crude protein extract on day 13.5 (lanes 2 and 7) were applied. After the phenyl-Sepharose purification step, partially purified samples were applied (strain BLx^T, lanes 2 and 6; compost sample, lanes 4 and 8). N-terminal amino acid sequences were determined for the bands indicated by arrowheads. (B) Comparisons of the N-terminal amino acid sequences in gelatinases from strain BLx^T and the compost sample on day 13.5 and between these gelatinases and bacillopeptidase F protein. Symbols: *, identical amino acid residues; :, positive residues defined by BLAST homology search analysis through the DNA Data Bank of Japan website (BLOSUM62 was selected as a scoring matrix for probability calculation of amino acid substitution); , unidentified residue.

FIG. 5.

Changes in the abundance ratio of strain BTa and amylase activity during the composting process. (A) The abundance ratio of strain BTa is represented as the DNA mass ratio (the DNA mass of strain BTa per DNA mass of the compost sample). The DNA mass of strain BTa was obtained from quantitative real-time PCR-targeted 16S rDNA from strain BTa with a LightCycler system and genomic DNA from strain BTa. The error bars indicate standard errors (n ≥ 3). (B) Amylase activity was measured by the starch-iodometric method. The error bars indicate standard errors (n = 3).

FIG. 6.

Zymogram of amylase activities of the compost samples and strain BTa culture supernatant with soluble starch as a substrate. All samples applied to the zymograph contained equal amounts of amylase activity units as determined by the starch-iodometric assay. Lane 1, culture supernatant from strain BTa; lane 2, compost sample on day 4.5; lane 3, compost sample on day 16.5.