Functional Characterization and Localization of a Bacillus subtilis Sortase and Its Substrate and Use of This Sortase System To Covalently Anchor a Heterologous Protein to the B. subtilis Cell Wall for Surface Display
Abstract:Sortases catalyze the covalent anchoring of proteins to the cell surface on Gram-positive bacteria. Bioinformatic analysis suggests the presence of structural genes encoding sortases and their substrates in the Bacillus subtilis genome. In this study, a -lactamase reporter was fused to the cell wall anchoring domain from a putative sortase substrate, YhcR. Covalent anchoring of this fusion protein to the cell wall was confirmed by using the eight-protease-deficient B. subtilis strain WB800 as the host. Inacti… Show more
“…This conclusion is further supported by the observation that the signal appears to form a spiral around the outside of the cell (Fig. 5 c), resembling the distribution of a sortase-anchored protein observed in B. subtilis [ 70 ]. Fig.…”
BackgroundThe use of fossil fuels is no longer tenable. Not only are they a finite resource, their use is damaging the environment through pollution and global warming. Alternative, environmentally friendly, renewable sources of chemicals and fuels are required. To date, the focus has been on using lignocellulose as a feedstock for microbial fermentation. However, its recalcitrance to deconstruction is making the development of economic processes extremely challenging. One solution is the generation of an organism suitable for use in consolidated bioprocessing (CBP), i.e. one able to both hydrolyse lignocellulose and ferment the released sugars, and this represents an important goal for synthetic biology. We aim to use synthetic biology to develop the solventogenic bacterium C. acetobutylicum as a CBP organism through the introduction of a cellulosome, a complex of cellulolytic enzymes bound to a scaffold protein called a scaffoldin. In previous work, we were able to demonstrate the in vivo production of a C. thermocellum-derived minicellulosome by recombinant strains of C. acetobutylicum, and aim to develop on this success, addressing potential issues with the previous strategy.ResultsThe genes for the cellulosomal enzymes Cel9G, Cel48F, and Xyn10A from C. cellulolyticum were integrated into the C. acetobutylicum genome using Allele-Coupled Exchange (ACE) technology, along with a miniscaffoldin derived from C. cellulolyticum CipC. The possibility of anchoring the recombinant cellulosome to the cell surface using the native sortase system was assessed, and the cellulolytic properties of the recombinant strains were assayed via plate growth, batch fermentation and sugar release assays.ConclusionsWe have been able to demonstrate the synthesis and in vivo assembly of a four-component minicellulosome by recombinant C. acetobutylicum strains. Furthermore, we have been able to anchor a minicellulosome to the C. acetobutylicum cell wall by the use of the native sortase system. The recombinant strains display an improved growth phenotype on xylan and an increase in released reducing sugar from several substrates including untreated powdered wheat straw. This constitutes an important milestone towards the development of a truly cellulolytic strain suitable for CBP.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0526-x) contains supplementary material, which is available to authorized users.
“…This conclusion is further supported by the observation that the signal appears to form a spiral around the outside of the cell (Fig. 5 c), resembling the distribution of a sortase-anchored protein observed in B. subtilis [ 70 ]. Fig.…”
BackgroundThe use of fossil fuels is no longer tenable. Not only are they a finite resource, their use is damaging the environment through pollution and global warming. Alternative, environmentally friendly, renewable sources of chemicals and fuels are required. To date, the focus has been on using lignocellulose as a feedstock for microbial fermentation. However, its recalcitrance to deconstruction is making the development of economic processes extremely challenging. One solution is the generation of an organism suitable for use in consolidated bioprocessing (CBP), i.e. one able to both hydrolyse lignocellulose and ferment the released sugars, and this represents an important goal for synthetic biology. We aim to use synthetic biology to develop the solventogenic bacterium C. acetobutylicum as a CBP organism through the introduction of a cellulosome, a complex of cellulolytic enzymes bound to a scaffold protein called a scaffoldin. In previous work, we were able to demonstrate the in vivo production of a C. thermocellum-derived minicellulosome by recombinant strains of C. acetobutylicum, and aim to develop on this success, addressing potential issues with the previous strategy.ResultsThe genes for the cellulosomal enzymes Cel9G, Cel48F, and Xyn10A from C. cellulolyticum were integrated into the C. acetobutylicum genome using Allele-Coupled Exchange (ACE) technology, along with a miniscaffoldin derived from C. cellulolyticum CipC. The possibility of anchoring the recombinant cellulosome to the cell surface using the native sortase system was assessed, and the cellulolytic properties of the recombinant strains were assayed via plate growth, batch fermentation and sugar release assays.ConclusionsWe have been able to demonstrate the synthesis and in vivo assembly of a four-component minicellulosome by recombinant C. acetobutylicum strains. Furthermore, we have been able to anchor a minicellulosome to the C. acetobutylicum cell wall by the use of the native sortase system. The recombinant strains display an improved growth phenotype on xylan and an increase in released reducing sugar from several substrates including untreated powdered wheat straw. This constitutes an important milestone towards the development of a truly cellulolytic strain suitable for CBP.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0526-x) contains supplementary material, which is available to authorized users.
“…In silico analysis showed that pLS20 did not contain a sortase gene. The bacterial genomes of most bacilli contain two genes, yhcS and ywpE , encoding probably functional but nonessential sortases ( 31 , 32 ). Remarkably, the genome of B. subtilis strain 168 used in our studies contains a deletion affecting the first 81 to 82 codons of ywpE and its entire upstream gene.…”
Conjugation, the process by which a DNA element is transferred from a donor to a recipient cell, is the main horizontal gene transfer route responsible for the spread of antibiotic resistance and virulence genes. Contact between a donor and a recipient cell is a prerequisite for conjugation, because conjugative DNA is transferred into the recipient via a channel connecting the two cells. Conjugative elements encode proteins dedicated to facilitating the recognition and attachment to recipient cells, also known as mating pair formation. A subgroup of the conjugative elements is able to mediate efficient conjugation during planktonic growth, and mechanisms facilitating mating pair formation will be particularly important in these cases. Conjugative elements of Gram-negative bacteria encode conjugative pili, also known as sex pili, some of which are retractile. Far less is known about mechanisms that promote mating pair formation in Gram-positive bacteria. The conjugative plasmid pLS20 of the Gram-positive bacterium Bacillus subtilis allows efficient conjugation in liquid medium. Here, we report the identification of an adhesin gene in the pLS20 conjugation operon. The N-terminal region of the adhesin contains a class II type thioester domain (TED) that is essential for efficient conjugation, particularly in liquid medium. We show that TED-containing adhesins are widely conserved in Gram-positive bacteria, including pathogens where they often play crucial roles in pathogenesis. Our study is the first to demonstrate the involvement of a class II type TED-containing adhesin in conjugation.
IMPORTANCE Bacterial resistance to antibiotics has become a serious health care problem. The spread of antibiotic resistance genes between bacteria of the same or different species is often mediated by a process named conjugation, where a donor cell transfers DNA to a recipient cell through a connecting channel. The first step in conjugation is recognition and attachment of the donor to a recipient cell. Little is known about this first step, particularly in Gram-positive bacteria. Here, we show that the conjugative plasmid pLS20 of Bacillus subtilis encodes an adhesin protein that is essential for effective conjugation. This adhesin protein has a structural organization similar to adhesins produced by other Gram-positive bacteria, including major pathogens, where the adhesins serve in attachment to host tissues during colonization and infection. Our findings may thus also open novel avenues to design drugs that inhibit the spread of antibiotic resistance by blocking the first recipient-attachment step in conjugation.
“…The host cell used in this study was B. subtilis WB800 strain [ 21 ], which was a gift from Dr. Sui-Lam Wong, University of Calgary, Canada. It contains the deletion of eight extracellular proteases, including nprE , aprE , epr , bpr , mpr , nprB , Δvpr, and wprA [ 20 ].…”
Ciona molecule against microbes-A24 (CiMAM) isolated from the marine chordate Ciona intestinalis is an antimicrobial peptide. To generate CiMAM-expressing transgenic Bacillus subtilis, we constructed a plasmid expressing recombinant CiMAM (rCiMAM) and introduced it into B. subtilis. Transgenic strains C117 and C166 were selected since they were able to highly and stably express rCiMAM. We studied the bactericidal activity of pepsin-digested extracts from rCiMAM-expressing strains against freshwater and euryhaline pathogens that commonly occur in aquaculture ponds and found no difference from that of lactoferricin-expressing strains. The bactericidal activity of 1-mL aliquot from a total 5.5 mL extracted from 5 mL of cultured C117 (1.45 × 108 CFU·mL−1) and C166 (2.17 × 108 CFU·mL−1) against halophilic bacteria was equivalent to the efficacy of 57.06 and 32.35 ng of Tetracycline against Vibrio natriegens, 47.07 and 25.2 ng against V. parahaemolyticus, and 58.17 and 36.55 ng against V. alginolyticus, respectively, indicating higher bactericidal activity of pepsin-extracts from rCiMAM-containing strains against halophilic bacteria compared to that from lactoferricin-containing strains. Since the antibacterial activity of rCiMAM-expressing B. subtilis strains shows higher competence against halophilic pathogens compared to that against freshwater and euryhaline pathogens, these strains are promising candidates to protect marine fish and shellfish from halophilic bacterial infection.
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