Antimicrobial Protein Candidates from the Thermophilic Geobacillus sp. Strain ZGt-1: Production, Proteomics, and Bioinformatics Analysis

Abstract

A thermophilic bacterial strain, Geobacillus sp. ZGt-1, isolated from Zara hot spring in Jordan, was capable of inhibiting the growth of the thermophilic G. stearothermophilus and the mesophilic Bacillus subtilis and Salmonella typhimurium on a solid cultivation medium. Antibacterial activity was not observed when ZGt-1 was cultivated in a liquid medium; however, immobilization of the cells in agar beads that were subjected to sequential batch cultivation in the liquid medium at 60 °C showed increasing antibacterial activity up to 14 cycles. The antibacterial activity was lost on protease treatment of the culture supernatant. Concentration of the protein fraction by ammonium sulphate precipitation followed by denaturing polyacrylamide gel electrophoresis separation and analysis of the gel for antibacterial activity against G. stearothermophilus showed a distinct inhibition zone in 15-20 kDa range, suggesting that the active molecule(s) are resistant to denaturation by SDS. Mass spectrometric analysis of the protein bands around the active region resulted in identification of 22 proteins with molecular weight in the range of interest, three of which were new and are here proposed as potential antimicrobial protein candidates by in silico analysis of their amino acid sequences. Mass spectrometric analysis also indicated the presence of partial sequences of antimicrobial enzymes, amidase, and dd-carboxypeptidase.

Keywords: Geobacillus; SDS-resistant proteins; antimicrobial proteins; cell-recycling; food spoilage bacteria; immobilization; thermophile.

Figure 1

Antibacterial activity of Geobacillus sp. strain ZGt-1 against (a) G. stearothermophilus strain 10; (b) B. subtilis; and (c) S. typhimurium CCUG 31969. The arrows are pointing to the inhibition zone.

Figure 2

The antibacterial activity of the cell-free supernatant obtained by sequential batch cultivation of the immobilized cells of Geobacillus sp. ZGt-1 against G. stearothermophilus strain 10 at 60 °C, at the end of (a) cycle # 1; (b) cycle # 5; (c) cycles # 11, 12, and 13; (d) cycles # 14, 15, 16, and 17; (e) cycles # 24 and 25. The yellow dot denotes the site where the supernatant was spotted; and (f) Summary of the antibacterial activity over the 25 cycles, the number of + symbols representing increasing degree of antibacterial activity.

Scheme 1

Workflow for identification of potential antimicrobial protein candidates in Geobacillus sp. ZGt-1: Desalted protein extract from Geobacillus sp. ZGt-1 was fractionated by SDS-PAGE, followed by detection of antibacterial activity on the gel against test microorganism (M.O.) and analysis of the active zone by mass spectrometry. S: sample, M: protein marker in the SDS-PAGE cartoons in the scheme. The experimental details are provided in the text.

Figure 3

Analysis of the antibacterial activity of the desalted protein fraction isolated from the culture supernatant produced by Geobacillus sp. ZGt-1 at 60 °C. The test organism was G. stearothermophilus strain 10. After SDS-PAGE separation of the protein in duplicates, the gel was divided into two, one part stained with Coomassie Brilliant Blue R 250 (a) and the other used for antibacterial assay (b). (a) Image of the SDS-PAGE separated protein fraction. Lane 1: Precision Plus Protein All Blue standards; Lane 2: Desalted protein fraction; (b) Antibacterial activity of the desalted protein extract after separating it on SDS-PAGE separated protein; the gel strip was placed in a Petri dish and covered with soft agar layer seeded with strain 10 and incubated at 60 °C. The white arrow is pointing to the inhibition zone ascribed to the antibacterial activity of the protein fraction. The inhibition zone corresponded to 15–20 kDa.