Lipolytic activity in Geobacillus thermoleovorans GE7: molecular and proteomic charecterization
Tlou, Matsobane Godfrey
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Geobacillus thermoleovorans GE-7 was isolated from the West-Driefontein goldmine in South Africa. It is a Gram-positive rod showing optimal growth at 65°C. This isolate was found to be able to grow on olive oil as the sole carbon source and on a variety of other lipid substrates, a feature which indicated that the bacterium produces lipases. In 2004, studies aimed at elucidating factors that would improve lipase production by G. thermoleovorans revealed that when the bacterium was cultured in medium optimized for lipase production, a production profile characterized by two enzyme activity peaks was observed. In 2005, a lipase (LipA) open reading frame (1251 bp) was amplified from the bacterium’s genome. Furthermore, lipase purification studies from the GE-7 culture grown in media containing olive oil resulted in the purification of a lipase (~45 kDa), which corresponded to the LipA ORF that had been amplified from the GE-7 genome. However, lipase activity staining with the supernatant from GE-7 cultured in media supplemented with olive oil revealed the presence of two lipolytic protein bands of different sizes. These observations and reports on the purification of a smaller lipase in addition to LipA from the culture supernatant of related Geobacilli led to the hypothesis that the two lipase production peaks observed with GE-7 represent two distinct lipases differentially expressed by the bacterium. The aim of this study was to characterize the lipolytic activity from this bacterium using molecular and proteomic techniques. The primary objective was to identify from GE-7 a lipase different from LipA and to profile its expression relative to that of LipA. Three authors had already reported on the purification of a smaller lipase (>20 kDa), however only two authors had published sequence information in this regard (Schmidt-Dannert et al., 1994; Lee et al., 2001). Since bacterial lipolytic enzyme families are characterized by very high intra-family sequence similarity, the published N-terminal amino acid sequences were used for similarity searches on the database to identify homologs of this protein from related Geobacilli. Similarity searches revealed that the published sequences shared very high similarity with a hypothetically conserved protein (HCP) from G. kaustophilus and no homology to any other known lipase. The nucleotide sequence of the HCP was used to design primers to facilitate PCR amplification of the homolog from GE-7. The “small lipase” ORF (440 bp) was amplified from the GE-7 genome, however, functional expression using tributyrate/olive oil-Rhodamine plate assays revealed that the protein was not lipolytic. Furthermore, secondary structure predictions using the “small lipase” ORF revealed that the sequence shared significant identity to the type II secretory pathway pseudopilin protein from related Bacillus species contrary to what was reported by the two authors. As a result of the above-mentioned findings a protein purification strategy aimed at isolating a lipase different from LipA was pursued. Since LipA was purified in a previous study from the second lipase peak and given that it was hypothesized that the first peak indicated the production of a lipase different from LipA, GE-7 was cultured, lipase production monitored and the cells harvested prior to the onset of the second peak. Lipase purification experiments from the culture supernatant corresponding to the second peak resulted in the isolation of a ~45 kDa protein. The protein band was analyzed by peptide mass fingerprinting and the amino acid sequence determined. Sequence analysis revealed that the protein that was purified from the first production peak is LipA, which served as an indication that LipA was produced at both peaks. GE-7 was cultured in media with (induced) and without (uninduced) the lipid substrate (olive oil) and the lipase production profiles compared. It was observed that under induced conditions the two production peaks persisted while only one peak was observed in the uninduced. However, reverse transcription-mediated PCR on RNA isolated from GE-7 cultured as described above with primers specific for lipA revealed that LipA is produced under both culture conditions. Moreover, LipA is present at both production peaks under induced conditions which supports the purification results. Detection of LipA from the uninduced culture indicates the GE-7 is constitutively expressed, contrary to the previous reports. Bioreactor studies aimed at relating the effect of media components on lipase production by GE- 7 revealed that, under induced conditions, significant lipase production is only observed after the depletion of glucose and that the second activity peak is only observed upon the uptake of the free fatty acids by the cells. This observation suggested the fatty acids could act as a signal for further lipase production which was supported by the one lipase production peak observed under uninduced conditions (no fatty acids in the culture medium). The observation that significant lipase production was observed at the stationary phase when the glucose concentration became limiting served as a possible indication of a mechanism of gene expression regulation known as catabolite repression. Which suggests that lipA could be down-regulated in the presence of simple sugars (glucose) and up-regulated upon depletion of glucose to afford the bacterium the ability to utilize olive oil as a carbon source. A conserved sequence (CRE-box) has been identified on the promoter regions of a number of genes that are subject to this mode of regulation. As a result, the lipA promoter region was amplified and sequenced. Analysis of the sequence revealed the presence of the CRE-box. Experiments were conducted to investigate whether catabolite repression was a possibility for the GE-7 lipA. GE-7 was cultured under induced conditions, lipase activity monitored and the cells spiked with additional glucose at the onset of the stationary phase. A significant drop in the lipase production was observed subsequent to the spike. The above-mentioned observations and experiments where olive oil (used as a sole carbon source) or stearic acid was used as the inducer revealed that changing certain components of the media changed the lipase production profile. All the findings in this study suggest that LipA is the only lipase produced under the culture conditions investigated and that the changes in the production profile could be due to the regulation of the gene in response to changes in the culture medium.