Abstract: An active area of research in the development of Streptococcus gordonii for use as a bacterial commensal vector involves the identification and utilization of strong promoters for high-level expression of heterologous products. Escherichia coli plasmid vectors containing different streptococcal promoters often fail to become established in E. coli for unknown reasons. Therefore, it is desirable at times to transform S. gordonii, which is naturally competent, with small quantities of nascently ligated DNA without using E. coli first to amplify or screen the product. By comparing the efficiency of two methods used to induce competence in S. gordonii, it was shown that the use of a synthetic competence stimulating peptide substantially enhanced plasmid uptake by S. gordonii. We amplified the amylase-binding protein (abpA) promoter from the S. gordonii genome and, using a synthetic peptide to induce competence, directly introduced plasmid DNA containing this promoter into S. gordonii as an unamplified product of ligation. This plasmid facilitated abundant secretion of a heterologous product by S. gordonii. By assessing the levels of heterologous product secreted by two plasmid constructs, it was possible to evaluate the relative strength of two native promoters.
Key words: Streptococcus gordonii, promoter, amylase binding protein, AbpA, expression, secretion, Gram positive, antigen, transformation, peptide, competence.
Resume : L'identification et l'utilisation de promoteurs forts permettant d'exprimer une grande quantite des proteines heterologues est un domaine de recherche actif dans la perspective d'utiliser Streptococcus gordonii comme vecteur bacterien commensal. Des vecteurs plasmidiques contenant differents promoteurs du streptocoque n'arrivent souvent pas a s'etablir chez Escherichia coli pour des raisons inconnues. Il est ainsi souhaitable de transformer S. gordonii qui est naturellement competent, avec de petites quantites d'ADN ligature sans utiliser d'abord E. coli pour amplifier ou cribler le produit. En comparant l'efficacite de deux methodes pour induire la competence chez S. gordonii, l'on a montre que l'utilisation de peptides synthetiques de stimulation de la competence augmentait substantiellement la captation du plasmide par S. gordonii. Nous avons amplifie le promoteur de abpA (amylase-binding protein) du genome de S. gordonii et, en utilisant un peptide synthetique pour induire la competence, nous avons directement introduit l'ADN plasmidique contenant ce promoteur dans S. gordonii en tant que produit non amplifie de la ligation. Ce plasmide a facilite une secretion abondante d'un produit heterologue par S. gordonii. En estimant les quantites de produit heterologue secrete par deux constructions plasmidiques, il a ete possible d'evaluer la force relative de deux promoteurs natifs.
Mots-cles : Streptococcus gordonii, promoteur, proteine liant l'amylase, AbpA, expression, secretion, Gram positif, antigene, transformation, peptide, competence.
[Traduit par la Redaction]
Introduction
Streptococcus gordonii, a human commensal bacterium, is being developed as a vaccine vector due to its ability to elicit mucosal and systemic immune responses (Wilson and Hruby 2005). Additionally, it may be useful as a vector for delivery of therapeutic molecules (Ricci et al. 2003), and has been used for protein production due to its ability to secrete soluble proteins into the culture supernatant (Lee et al. 2002; Warren et al. 2005). These applications all require high-level expression of heterologous products, which can be facilitated through the use of native promoters and secretion signal sequences situated on multi-copy plasmids. An active area of research involves the identification and evaluation of the strength of native S. gordonii promoters (Provvedi et al. 2005).
Under glucose-deficient culture conditions, S. gordonii abundantly secretes a 20.5 kDa amylase-binding protein (AbpA) (Rogers et al. 1998). Amylase, the most abundant enzyme in human saliva, catalyzes the hydrolysis of [alpha]-1,4-glucosidic linkages in starch. Based on observations that amylase-binding streptococci appear to be restricted to animal hosts that secrete salivary amylase, it has been theorized that the binding of amylase by oral streptococci may be critical for their ability to colonize and persist in the host (Scannapieco et al. 1994; Brown et al. 1999). Additionally, the binding of amylase may facilitate bacterial growth by providing fermentable carbohydrates from dietary starch. Analysis of the abpA sequence reveals a putative catabolic response element (CRE) 153 bp downstream of the start codon (Rogers and Scannapieco 2001). This CRE may participate in transcriptional repression of abpA in the presence of saccharides, when starch metabolism is not necessitated (Rogers and Scannapieco 2001). Rogers and Scannapieco (2001) identified the transcriptional start site of abpA, but the genomic sequence 5' to this region remains uncharacterized.
A potential drawback to evaluating promoter strengths for purposes of expression by S. gordonii is that Escherichia coli--into which plasmid constructs are routinely subcloned, screened, and amplified--does not stably maintain high-copy vectors containing particular streptococcal promoters (Stassi et al. 1982; Provvedi et al. 2005). For this reason, it is desirable at times to be able to transform ligated DNA containing potentially strong promoters directly into S. gordonii without subcloning into E. coli. Even though S. gordonii is naturally competent, the efficiency with which DNA is introduced using traditional methodologies (that is by inducing competence with calf or horse serum) does not permit the routine introduction of small quantities of DNA such as the product in typical ligation reactions.
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