Inhibition of the growth of Bacillus subtilis DSM10 by a newly discovered antibacterial protein from the soil metagenome

Abstract

A functional metagenomics based approach exploiting the microbiota of suppressive soils from an organic field site has succeeded in the identification of a clone with the ability to inhibit the growth of Bacillus subtilis DSM10. Sequencing of the fosmid identified a putative β-lactamase-like gene abgT. Transposon mutagenesis of the abgT gene resulted in a loss in ability to inhibit the growth of B. subtilis DSM10. Further analysis of the deduced amino acid sequence of AbgT revealed moderate homology to esterases, suggesting that the protein may possess hydrolytic activity. Weak lipolytic activity was detected; however the clone did not appear to produce any β-lactamase activity. Phylogenetic analysis revealed the protein is a member of the family VIII group of lipase/esterases and clusters with a number of proteins of metagenomic origin. The abgT gene was sub-cloned into a protein expression vector and when introduced into the abgT transposon mutant clones restored the ability of the clones to inhibit the growth of B. subtilis DSM10, clearly indicating that the abgT gene is involved in the antibacterial activity. While the precise role of this protein has yet to fully elucidated, it may be involved in the generation of free fatty acid with antibacterial properties. Thus functional metagenomic approaches continue to provide a significant resource for the discovery of novel functional proteins and it is clear that hydrolytic enzymes, such as AbgT, may be a potential source for the development of future antimicrobial therapies.

Keywords: antibacterial activity; bacillus subtilis; functional metagenomics; β-lactamase-like gene.

Figure 1.

Screening of the soil metagenomic library. Metagenomic clones were overlaid with soft agar containing B. subtilis DSM10. Arrow indicates clone TO-T 020 P12 producing a zone of growth inhibition.

Figure 2.

Schematic representation of putative open reading frames. Putative open reading frames were identified in the 35277 bp DNA insert of fosmid clone TO-T-020-P12. The arrows represent the position and direction of transcription of each open reading. The numbers correspond to the ORFs listed in Table 1. The abgT gene (ORF 26 -Table 1) is highlighted in a darker color . Arrows containing * represent partial open reading frames.

Figure 3.

Phylogenetic analysis of the deduced AbgT amino acid sequence. The tree contains proteins closely related to AbgT, putative β-lactamases, Class C β-lactamases and lipase family members. The tree represents the Family VIII lipase cluster and is rooted to the other lipase family groups.

Figure 4.

Lipolytic and antibacterial activity of the abgT gene. (A) Clone TO-T-020-P12 produced weak lipolytic activity on agar plates containing 1% tributyrin. When the abgT gene was disrupted by Tn5 insertion in TO-T 020 P12 E2 lipolytic activity was lost. Metagenomic lipase Lpc53E1 was previously characterized in our laboratory and was used for comparison of lipolytic activity. (B) Random mutagenesis of fosmid TO-T 020 P12 produced 2 mutants unable to inhibit the growth of B. subtilis DSM10. Both mutants regained the ability to inhibit growth after complementation of the disrupted abgT gene. (1) TO-T- 020-P12 (2) TO-T-020-P12 E2 (3) TO-T-020-P12 F2 (4) TO-T-020-P12 with pBadMyc-HisA-AB-RH1 (5) TO-T-020-P12 E2 with pBadMyc-HisA-AB-RH1 (6) TO-T-020-P12 F2 with pBadMyc-HisA-AB-RH1.

Figure 5.

Purification of His₆-AbgT protein. His₆- AbgT was purified from the total soluble protein fraction of cell lysate of E coli EPI300^TM T1 transformed with the recombinant vector pBadMyc-HisA-AB-RH1. The expressed antibacterial protein was purified from the supernatant by Ni-NTA spin column and resolved by SDS-PAGE on a 10% gel. (A) Anti-bacillus activity of the purified Abg protein from cell lysate (1; purified fraction of Ni-NTA column, 2; protein fraction from cell lysate, 3; supernatant fraction, 4; wash from Ni-NTA elution, 5; control with cell free lysate from EP1300 cells). (B) Lane 1 stained with Invision His-tag In-gel stain showing the mass of the purified protein at approximately 50 kDa. (C) Lane 1 showing the purified protein stained with Coomassie brilliant blue.

Figure 6.

Partial alignment of AbgT with related members of the family VIII group of lipases. The alignment includes; closely related putative β-lactamase proteins from Turneriella parva, Asticcacaulis biprosthecum and Parvibaculum lavamentivorans; antibacterial protein from CcAb2; esterase and hydrolase enzymes from uncultured bacteria (EstM-N2 and Lpc53E1), Burkholderia gladioli, Brevibacteroum linens and Gordonia sp; class C β-lactamases from Escherichia coli and Enterobacter cloacae. Conserved sites S-X-X-K and Y are shaded. The numbers represent amino acids of the AbgT protein.