Improved Vectors for Nisin-Controlled Expression in Gram-Positive Bacteria

Bryan, Edward M.; Bae, Taeok; Kleerebezem, Michiel; Dunny, Gary M.

doi:10.1006/plas.2000.1484

Cited by 246 publications

(147 citation statements)

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“…Prior to the microarray analysis, both DclpP and DclpX were complemented by providing, respectively, the full-length clpP and clpX genes in trans using the nisin-inducible plasmid pMSP3535 (Bryan et al, 2000). Several phenotypes characteristic of the DclpP and DclpX strains (Kajfasz et al, 2009), including slow growth rates and aggregation in broth, were fully reversed by the complementation (Supplementary Figs S1, S2 and S3).…”

Section: Resultsmentioning

confidence: 99%

Transcriptome analysis reveals that ClpXP proteolysis controls key virulence properties of Streptococcus mutans

2011

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The ClpXP proteolytic complex is critical for maintaining cellular homeostasis, as well as expression of virulence properties. However, with the exception of the Spx global regulator, the molecular mechanisms by which the ClpXP complex exerts its influence in Streptococcus mutans are not well understood. Here, microarray analysis was used to provide novel insights into the scope of ClpXP proteolysis in S. mutans. In a DclpP strain, 288 genes showed significant changes in relative transcript amounts (P¡0.001, twofold cut-off) as compared with the parent. Similarly, 242 genes were differentially expressed by a DclpX strain, 113 (47 %) of which also appeared in the DclpP microarrays. Several genes associated with cell growth were downregulated in both mutants, consistent with the slow-growth phenotype of the Dclp strains. Among the upregulated genes were those encoding enzymes required for the biosynthesis of intracellular polysaccharides (glg genes) and malolactic fermentation (mle genes). Enhanced expression of glg and mle genes in DclpP and DclpX strains correlated with increased storage of intracellular polysaccharide and enhanced malolactic fermentation activity, respectively. Expression of several genes known or predicted to be involved in competence and mutacin production was downregulated in the Dclp strains. Follow-up transformation efficiency and deferred antagonism assays validated the microarray data by showing that competence and mutacin production were dramatically impaired in the Dclp strains. Collectively, our results reveal the broad scope of ClpXP regulation in S. mutans homeostasis and identify several virulencerelated traits that are influenced by ClpXP proteolysis. INTRODUCTIONStreptococcus mutans is a member of the oral microbiome known for its close association with dental caries and, occasionally, infective endocarditis. The niche in which S. mutans thrives is the biofilm that forms on the enamel surface of teeth (Loesche, 1986). The dental biofilm environment is constantly and unpredictably changing due to the eating habits of the human host, resulting in large fluctuations in nutrient source and availability, pH, and oxygen tension, among other stresses . The remarkable ability of S. mutans to tolerate and thrive during stressful conditions, particularly low pH, is closely linked to its virulence in the oral cavity.The Clp proteolytic complex is critical in maintaining cellular homeostasis, particularly for organisms that must continually endure environmental fluctuations (Frees et al., 2004;Gottesman, 2003;Jenal & Hengge-Aronis, 2003; Kajfasz et al., 2009). In S. mutans, Clp proteases are the result of the association of the ClpP peptidase with one of several Clp ATPases (ClpC, ClpE or ClpX), forming barrelshaped complexes that will target proteins for degradation (Kajfasz et al., 2009;Lemos & Burne, 2002). Although S. mutans also encodes ClpB and ClpL ATPases, these proteins do not contain the recognition tripeptide that permits interaction with ClpP, and are believed to function mainly...

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Section: Resultsmentioning

confidence: 99%

Transcriptome analysis reveals that ClpXP proteolysis controls key virulence properties of Streptococcus mutans

2011

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“…The nisin promoter (P nisA ) cassette containing an erythromycin selection marker (erm) was PCR-amplified using the AccuPrime DNA Taq Polymerase High Fidelity kit (Invitrogen) with primer set PNISaF/PNISR (see Table 2 for primer specifications) from plasmid pMSP3535 (Bryan et al, 2000), a kind gift from Dr B. Buttaro. PCRs were performed in 30 ml reaction mixtures containing 2 ml template DNA, 2 ml (20 pmol) forward primer, 2 ml (20 pmol) reverse primer, 21.5 ml distilled H 2 O, 2 ml buffer (provided by the manufacturer) and 0.5 ml AccuPrime DNA Taq polymerase.…”

Section: Methodsmentioning

confidence: 99%

Genetic modifications to temperate Enterococcus faecalis phage ϕEf11 that abolish the establishment of lysogeny and sensitivity to repressor, and increase host range and productivity of lytic infection

et al. 2013

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“…The 1278 bp coPAI gene (codon-optimized Propionibacterium acnes isomerase in a plant vector; BASF), encoding the linoleic acid isomerase from P. acnes (Hornung et al, 2005), was amplified and cloned into the nisin-inducible lactococcal plasmid pNZ8048 (containing the P nisA promoter) as described previously (Rosberg-Cody et al, 2007). The coPAI gene was excised from pNZ8048-coPAI using PstI and XbaI restriction enzymes and ligated into the same sites of the Lactobacillus nisin-inducible plasmid pMSP3535 (Bryan et al, 2000), as described by the supplier (New England Biolabs), resulting in the construct pEcoPAIL (Fig. 1).…”

Section: Methodsmentioning

confidence: 99%

Recombinant lactobacilli expressing linoleic acid isomerase can modulate the fatty acid composition of host adipose tissue in mice

Rosberg-Cody

Stanton

O’Mahony³

et al. 2011

Microbiology

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We have previously demonstrated that oral administration of a metabolically active Bifidobacterium breve strain, with ability to form cis-9, trans-11 conjugated linoleic acid (CLA), resulted in modulation of the fatty acid composition of the host, including significantly elevated concentrations of c9, t11 CLA and omega-3 (n-3) fatty acids in liver and adipose tissue. In this study, we investigated whether a recombinant lactobacillus expressing linoleic acid isomerase (responsible for production of t10, c12 CLA) from Propionibacterium acnes (PAI) could influence the fatty acid composition of different tissues in a mouse model. Linoleic-acid-supplemented diets (2 %, w/w) were fed in combination with either a recombinant t10, c12 CLA-producing Lactobacillus paracasei NFBC 338 (Lb338), or an isogenic (vector-containing) control strain, to BALB/c mice for 8 weeks. A third group of mice received linoleic acid alone (2 %, w/w). Tissue fatty acid composition was assessed by GLC at the end of the trial. Ingestion of the strain expressing linoleic acid isomerase was associated with a 4-fold increase (P,0.001) in t10, c12 CLA in adipose tissues of the mice when compared with mice that received the isogenic non-CLA-producing strain. The livers of the mice that received the recombinant CLA-producing Lb338 also contained a 2.5-fold (albeit not significantly) higher concentration of t10, c12 CLA, compared to the control group. These data demonstrate that a single gene (encoding linoleic acid isomerase) expressed in an intestinal microbe can influence the fatty acid composition of host fat. INTRODUCTIONEvidence is emerging to support the concept that the enteric microbiota can exert profound effects on human health and disease, involving complex host-bacteria interactions that are as yet poorly understood. The gut microbiota is important to the host with regard to metabolic functions, providing nutrients and conferring an ability to resist bacterial infections (Marchesi & Shanahan, 2007;Wilks, 2007), as well as playing a dominant role in the education of the intestinal mucosal immune responses. Furthermore, the enteric microbiota has been shown to exert effects on disease processes outside the gut, including an impact on obesity and non-alcoholic fatty liver disease (Dumas et al., 2006;Marchesi & Shanahan, 2007).Conjugated linoleic acid (CLA) is a beneficial bacterial metabolite formed via microbial isomerization of linoleic acid. CLA is a collective term describing different isomers of linoleic acid with conjugated double bonds, the cis-9, trans-11 (c9, t11) CLA isomer being the most abundant form and the t10, c12 CLA isomer accounting for~1 % of total milk fat CLA (Jensen, 2002). It has been reported that t10, c12 CLA is the most potent isomer in terms of potential to prevent cell proliferation and induce apoptosis in cancer cells (Cho et al., 2005(Cho et al., , 2006Kim et al., 2002a; Lee et al., 2006b; Ochoa et al., 2004). t10, c12 CLA is also associated with decreased body fat and increased lean body mass in various animal...

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Improved Vectors for Nisin-Controlled Expression in Gram-Positive Bacteria

Cited by 246 publications

References 20 publications

Transcriptome analysis reveals that ClpXP proteolysis controls key virulence properties of Streptococcus mutans

Transcriptome analysis reveals that ClpXP proteolysis controls key virulence properties of Streptococcus mutans

Genetic modifications to temperate Enterococcus faecalis phage ϕEf11 that abolish the establishment of lysogeny and sensitivity to repressor, and increase host range and productivity of lytic infection

Recombinant lactobacilli expressing linoleic acid isomerase can modulate the fatty acid composition of host adipose tissue in mice

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