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We investigated spatial and temporal variations in bacterial community structures as well as the presence of three functional proteolytic enzyme genes in the sediments of a hypereutrophic freshwater lake in order to acquire an insight into dynamic links between bacterial community structures and proteolytic functions. Bacterial communities determined from 16S rRNA gene clone libraries markedly changed bimonthly, rather than vertically in the sediment cores. The phylum Firmicutes dominated in the 4–6 cm deep sediment layer sample after August in 2007, and this correlated with increases in interstitial ammonium concentrations (p < 0.01). The Firmicutes clones were mostly composed of the genus Bacillus. npr genes encoding neutral metalloprotease, an extracellular protease gene, were detected after the phylum Firmicutes became dominant. The deduced Npr protein sequences from the retrieved npr genes also showed that most of the Npr sequences used in this study were closely related to those of the genus Bacillus, with similarities ranging from 61% to 100%. Synchronous temporal occurrences of the 16S rRNA gene and Npr sequences, both from the genus Bacillus, were positively associated with increases in interstitial ammonium concentrations, which may imply that proteolysis by Npr from the genus Bacillus may contribute to the marked increases observed in ammonium concentrations in the sediments. Our results suggest that sedimentary bacteria may play an important role in the biogeochemical nitrogen cycle of freshwater lakes.