Selection and characterization of a promoter for expression of single-copy recombinant genes in Gram-positive bacteria

Provvedi, Roberta; Maggi, Tiziana; Oggioni, Marco R.; Manganelli, Riccardo; Pozzi, Gianni

doi:10.1186/1472-6750-5-3

Cited by 3 publications

(2 citation statements)

References 36 publications

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“…The published SF370 genome does not contain promoter annotations, so we examined the entire genome in 100,000-bp segments (available for download at ftp://ftp.genome.ou.edu/pub/strep) and used the Vector NTI advance 9.0 sequence analysis suite (Invitrogen) to identify sequences that were similar (75% similarity threshold) to consensus streptococcal promoter sequences [64]. We cross-referenced the clusters containing a single upstream putative promoter sequence with a list of rho-independent terminator sequences, previously identified in the SF370 genome by TransTerm (www.tigr.org/software/transterm.html).…”

Section: Methodsmentioning

confidence: 99%

Novel Algorithms Reveal Streptococcal Transcriptomes and Clues about Undefined Genes

et al. 2007

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Bacteria–host interactions are dynamic processes, and understanding transcriptional responses that directly or indirectly regulate the expression of genes involved in initial infection stages would illuminate the molecular events that result in host colonization. We used oligonucleotide microarrays to monitor (in vitro) differential gene expression in group A streptococci during pharyngeal cell adherence, the first overt infection stage. We present neighbor clustering, a new computational method for further analyzing bacterial microarray data that combines two informative characteristics of bacterial genes that share common function or regulation: (1) similar gene expression profiles (i.e., co-expression); and (2) physical proximity of genes on the chromosome. This method identifies statistically significant clusters of co-expressed gene neighbors that potentially share common function or regulation by coupling statistically analyzed gene expression profiles with the chromosomal position of genes. We applied this method to our own data and to those of others, and we show that it identified a greater number of differentially expressed genes, facilitating the reconstruction of more multimeric proteins and complete metabolic pathways than would have been possible without its application. We assessed the biological significance of two identified genes by assaying deletion mutants for adherence in vitro and show that neighbor clustering indeed provides biologically relevant data. Neighbor clustering provides a more comprehensive view of the molecular responses of streptococci during pharyngeal cell adherence.

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Section: Methodsmentioning

confidence: 99%

Novel Algorithms Reveal Streptococcal Transcriptomes and Clues about Undefined Genes

et al. 2007

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“…Most often, the construction of plasmid-less Bacillus strains is performed by homologous recombination-mediated integration of the desired genes into the bacterial chromosome [ 10 ]. In some instances, specialized site-specific recombination [ 27 ] and transposition [ 28 , 29 ] are used for the same integrative purposes.…”

Section: Introductionmentioning

confidence: 99%

Overproduction of Bacillus amyloliquefaciens extracellular glutamyl-endopeptidase as a result of ectopic multi-copy insertion of an efficiently-expressed mpr gene into the Bacillus subtilis chromosome

et al. 2011

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BackgroundPlasmid-less, engineered Bacillus strains have several advantages over plasmid-carrier variants. Specifically, their stability and potential ecological safety make them of use in industrial applications. As a rule, however, it is necessary to incorporate many copies of a key gene into a chromosome to achieve strain performance that is comparable to that of cells carrying multiple copies of a recombinant plasmid.ResultsA plasmid-less B. subtilis JE852-based strain secreting glutamyl-specific protease (GSP-the protein product of the mpr gene from B. amyloliquefaciens) was constructed that exhibits decreased levels of other extracellular proteases. Ten copies of an mprB.amy cassette in which the GSP gene was placed between the promoter of the B. amyloliquefaciens rplU-rpmA genes and the Rho-independent transcription terminator were ectopically inserted into designated (3 copies) and random (7 copies) points in the recipient chromosome. The resulting strain produced approximately 0.5 g/L of secreted GSP after bacterial cultivation in flasks with starch-containing media, and its performance was comparable to an analogous strain in which the mprB.amy cassette was carried on a multi-copy plasmid.ConclusionA novel strategy for ectopically integrating a cassette into multiple random locations in the B. subtilis chromosome was developed. This new method is based on the construction of DNA fragments in which the desired gene, marked by antibiotic resistance, is sandwiched between "front" and "back" portions of random chromosomal DNA restriction fragments. These fragments were subsequently inserted into the targeted sites of the chromosome using double-cross recombination. The construction of a marker-free strain was achieved by gene conversion between the integrated marked gene and a marker-less variant carried by plasmid DNA, which was later removed from the cells.

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Comparison of transformation protocols in Streptococcus gordonii and evaluation of native promoter strength using a multiple-copy plasmid

et al. 2007

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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.

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Selection and characterization of a promoter for expression of single-copy recombinant genes in Gram-positive bacteria

Cited by 3 publications

References 36 publications

Novel Algorithms Reveal Streptococcal Transcriptomes and Clues about Undefined Genes

Novel Algorithms Reveal Streptococcal Transcriptomes and Clues about Undefined Genes

Overproduction of Bacillus amyloliquefaciens extracellular glutamyl-endopeptidase as a result of ectopic multi-copy insertion of an efficiently-expressed mpr gene into the Bacillus subtilis chromosome

Comparison of transformation protocols in Streptococcus gordonii and evaluation of native promoter strength using a multiple-copy plasmid

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