Construction of vectors for inducible and constitutive gene expression in Lactobacillus

Abstract

SummaryMicroarray analysis of the genome of Lactobacillus acidophilus identified a number of operons that were differentially expressed in response to carbohydrate source or constitutively expressed regardless of carbohydrate source. These included operons implicated in the transport and catabolism of fructooligosaccharides (FOS), lactose (lac), trehalose (tre) and genes directing glycolysis. Analysis of these operons identified a number of putative promoter and repressor elements, which were used to construct a series of expression vectors for use in lactobacilli, based on the broad host range pWV01 replicon. A β‐glucuronidase (GusA3) reporter gene was cloned into each vector to characterize expression from each promoter. GUS reporter assays showed FOS, lac and tre based vectors to be highly inducible by their specific carbohydrate and repressed by glucose. Additionally, a construct based on the phosphoglycerate mutase (pgm) promoter was constitutively highly expressed. To demonstrate the potential utility of these vectors, we constructed a plasmid for the overexpression of the oxalate degradation pathway (Frc and Oxc) of L. acidophilus NCFM. This construct was able to improve oxalate degradation by L. gasseri ATCC 33323 and compliment a L. acidophilus oxalate‐deficient mutant. Development of these expression vectors could support several novel applications, including the expression of enzymes, proteins, vaccines and biotherapeutics by intestinal lactobacilli.

Directed Chromosomal Integration and Expression of the Reporter Gene gusA3 in Lactobacillus acidophilus NCFM

“…The GusA3 activity from the chromosomal integration strains was compared with activity from the pTRK892 plasmid (strain NCK1829), where gusA3 was under the control of a phosphoglycerate mutase (pgm) promoter, previously shown to have high, constitutive expression (3,8). The GusA3 activity of NCK2136 (slpA) was nearly 10-fold higher than the activity of NCK1829(pTRK892) in both glucose and lactose (Fig.…”

“…GusA3 activity was measured by the fluorescence produced upon hydrolysis of the substrate 4-methyl-umbelliferyl-␤-Dglucuronide (MUG). Assays were performed as described previously (8,30), with modifications. Cells were grown (10 ml) to log phase (optical density at 600 nm [OD 600 ] of 0.6 to 0.8) and collected by centrifugation at 1,717 ϫ g for 10 min at room temperature.…”

Evaluation of Phytate-Degrading Lactobacillus Culture Administration to Broiler Chickens

“…L. acidophilus NCFM and L. gasseri ATCC 33323 were originally isolated from the human gastrointestinal tract (61,62). These cultures are commonly used as model organisms in research investigating mechanisms of probiotic functionality because they are readily transformed (63,64) and genetically tractable (37,65) and because a complete genome sequences is available for these microorganisms (62,66). L. gallinarum was originally isolated from the crop of a chicken (67) and has been demonstrated to reduce gastrointestinal colonization of Campylobacter jejuni in experimentally challenged broiler chickens (8).…”

“…subtilis phyA was codon optimized and cloned into pTRK882, under the control of the constitutive high-expressing P pgm promoter from L. acidophilus NCFM (37), in order to maximize expression in Lactobacillus species. This expression system has been previously demonstrated to be effective in enzyme expression (37), the production and delivery of immune modulating cytokines (59), and an anthrax vaccine (60), and its wide host range allowed the transformation of L. acidophilus, L. gallinarum, and L. gasseri.…”

Directional Activation of Intestinal Dendritic Cells by Oral Targeted Multivalent Vaccine

“…Since Lactobacillus species thrive in the low pH of the stomach, using it as a vaccine delivery vehicle protects the vaccine subunit from the harsh acidic environment and protects the vaccine bioavailability. Meanwhile, researchers have also developed both inducible and constitutive expression vectors effective in lactobacilli species to deliver immunogenic antigens [1,[4][5][6]. Although immune tolerance to a commensal inhabitant of the gastrointestinal mucosa was an initial concern with this strategy certain lactobacilli such as L. gasseri, are able to overcome tolerance likely via strong innate adjuvant properties [7,8].…”