Differential Activities of FourLactobacillus caseiPromoters during Bacterial Transit through the Gastrointestinal Tracts of Human-Microbiota-Associated Mice

Abstract

In a previous study using fusion of the deregulated lactose promoter lacTp* and reporter genes, we suggested that Lactobacillus casei could initiate de novo protein synthesis during intestinal transit. In order to confirm this finding and extend it to other promoters, we adopted a reverse transcriptase quantitative PCR (RT-QPCR) approach combined with a transcriptional fusion system consisting of luciferase genes under the control of four promoters (ccpA, dlt, ldh, and lacT*) from L. casei DN-114 001. Promoter expression was monitored during cell growth, and variable luciferase activities were detected. In 3-day cultures, all the genetically modified strains survived but without exhibiting luciferase activity. Luciferase mRNA levels determined by RT-QPCR analysis (RNA/CFU) were not significant. The cultures were administered to human-microbiota-associated mice, and the feces were collected 6 h later. L. casei promoters lacTp* and ldhp initiated mRNA synthesis during gastrointestinal transit. The promoters, ccpAp and dltp, exhibited no luciferase activity, nor was de novosynthesized luciferase mRNA detected in the feces. L. casei seems to adapt its physiology to the gastrointestinal tract environment by modulating promoter activities. The approach (fecal transcriptional analysis) described herein may, moreover, be of value in studying gene expression of transiting bacteria in human fecal specimens.Lactobacillus casei has attracted considerable interest as a probiotic over the last few years (15,19). The strain L. casei DN-114 001 has been shown to alleviate acute diarrhea in healthy children (21,22), to modulate the production of proinflammatory cytokines in Crohn's disease ex vivo (3), and to enhance the immune system during bacterial gastrointestinal (GI) transit (20). However, little is known about the survival of L. casei species and their physiological status in the environment of the GI tract.Probiotics are usually ingested with fermented milk during the late stationary phase of the bacterial cycle. At the onset of the stationary phase, the overall rate of protein synthesis has been shown to drop precipitously to a rate equivalent to approximately 0.2% of the exponential growth rate (13). These changes are concomitant with a decrease in overall mRNA and DNA synthesis (13). The behavior of such metabolically inactive cells in the GI environment was thus investigated with a view to identifying the mechanisms underlying their action on health.To carry out this work, we developed a genetic approach using transcriptional fusion of luciferase genes (luxA and luxB) and selected promoters to study the response of lactic acid bacteria to the GI tract environment (5). The same approach was then extended to a probiotic microorganism, L. casei DN-114 001, to study its response to the GI tract in the humanmicrobiota-associated (HMA) mouse model using transcriptional fusion of the modified L. casei lacTEGF operon promoter and luxA and luxB genes from Photorhabdus luminescens. Resulting data suggest that L. casei can init...

Novel Luciferase Reporter System for In Vitro and Organ-Specific Monitoring of Differential Gene Expression inListeria monocytogenes

“…Moreover, the pPL2lux system allows direct, quantitative determination of luciferase activity and the corresponding promoter strength, intrinsically providing the ability to monitor promoter strength in a real-time fashion. This characteristic provides a major advantage compared with many of the reporter systems available to date for gram-positive bacteria, which typically require disruption of bacterial cells and/or addition of a substrate, followed by spectophotometric measurement to quantitatively determine enzyme and corresponding promoter activities (1,7,28,30,32).…”

“…Alternative strategies for both gram-negative and gram-positive bacteria have involved the luciferase-encoding luxAB genes, typically derived from Vibrio fischeri, Vibrio harveyi, or Photorhabdus luminescens (25). Although the level of the background bioluminescence is extremely low compared to the level of endogenous background fluorescence, luxAB systems require addition of an exogenous aldehyde as a substrate in the light emission reaction (17,28,30,33). This aldehyde requirement can be overcome by using the synthetic operon luxCD-ABE, as luxCDE encodes a fatty acid reductase complex involved in synthesis of the fatty aldehyde substrate for the bioluminescence reaction catalyzed by the luxAB-encoded luciferase (25).…”

Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118

“…We therefore attempted to express both the luciferase marker (lux) and gfp in Lactobacillus strains using pLS203 as a vector. The strategy of expressing lux has been used by colleagues to tag pathogens (3) and other commensal bacteria (45). A synthetic luxABCDE operon encoding luciferase (luxAB) and a fatty acid reductase complex (luxCDE) was obtained from pFT1 (a vector with the backbone of pUC19 containing the luxABCDE operon; I. Monk, unpublished data).…”

Expression and Secretion of Cholera Toxin B Subunit in Lactobacilli

“…We chose the LDH promoter not only because it is constitutively expressed in Lactobacillus species, 18) but also because this is one of the strongest and highest efficiency promoters known in the L. casei vector system. 19) pCTB was designed for intracellular expression of CTB in LAB. As shown in Fig.…”