The production of bioluminescent lactic acid bacteria suitable for the rapid assessment of starter culture activity in milk

Abstract

Promoter elements from Lactobacillus casei were isolated with an Escherichia coli promoter probe vehicle and inserted 5′ to the luxA/B genes from Vibrio fischeri located within a pCK1‐based shuttle vector. Three independent promoter‐lux constructs were each used to transform Lactobacillus casei, Lactococcus lactis and Lactococcus lactis subsp. diacetylactis by electroporation. Transformants of all three bacteria which expressed a bioluminescent phenotype in the presence of exogenous dodecanal were obtained. By virtue of monitoring changes in light production, these recombinant micro‐organisms could form the basis of a rapid monitoring system for antimicrobial substances in milk active against starter culture bacteria. In addition, the research potential of in vivo bioluminescence for monitoring gene expression in lactic acid bacteria in situ within fermentation systems can now be addressed.

Viable but non-culturable forms of potentially pathogenic bacteria in water

“…Freezethaw lysis of L. pneurnophila followed by DNA amplification detected both culturable and viable but non-culturable cells (Bej et al 1991). Escherichia coli detection by the PCR procedure, following enzymic lysis, corresponded to culturable cells as determined by plate counting (Bej et al 1990).…”

“…Photometry of light emitted by recombinant strains of bacteria which express luciferase and associated enzymes can monitor intracellular energy production (Ahmad & Stewart 1991;Ellison et al 1991). This novel and promising technique should be applicable to micro-organisms in aquatic environments although the oxidation of flavin mononucleotide by luciferase may represent an abnormal drain on the energy resources of cells.…”

“…If the sample does not contain DNA complementary to the primer sequences no amplification will occur. By choosing primer base sequences which are specific for a bacterial species the presence in a sample of the DNA from a single cell can be detected (Bej et al 1990).…”

The use of bacterial luciferase genes as reporter genes in Lactococcus: regulation of the Lactococcus lactis subsp. lactis lactose genes

“…Bacterial luciferase genes have been used widely as in vivo reporters of gene expression in Gram-negative bacteria and there are a growing number of examples of lux as a monitor in Gram-positive species Guijarro et al, 1988;Carmi et aZ., 1987;Ahmad & Stewart, 1991 ;Sohaskey et al, 1992). Light emission has several advantages over alternative systems, providing rapid assays of superior sensitivity without the need for cell disruption and resulting in real-time expression data (for reviews, see Meighen, 1988Meighen, , 1991Stewart & Williams, 1992).…”

Biotechnology-Based Methods for the Detection, Enumeration and Epidemiology of Food Poisoning and Spoilage Organisms

“…Inhibition of starter culture bacteria may be caused by antibiotics, detergents~disinfectants or bacteriophage, and although, in many cases, the precise identification of the inhibitory substance is not required, there is a need for a test which reports their presence more rapidly than the current and extensively used disc and dye reduction assays (Harrigan and McCance, 1976). Bioluminescent Lactobacillus easei has been used successfully in a prototype system to detect low levels of antibiotic activity within 60 min (Ahmad and Stewart, 1990). A bioluminescent starter culture assay system would be used as a test reagent, which would not itself be utilized for any manufacturing process but which could screen for any antimicrobial activity detrimental to culture growth and viability.…”