Development and validation of a species-independent functional gene microarray that targets lactic acid bacteria

Abstract

During the last few years, genome-related information has become available for many microorganisms, including important food-related bacteria. Lactic acid bacteria (LAB) are important industrially in the production of fermented foods such as dairy products, sausages, sourdoughs, and vegetables. Despite their limited metabolic capacity, LAB contribute considerably to important characteristics of fermented foods, such as flavor and texture. In the present study, a species-independent functional gene microarray was developed that targets 406 genes that play key roles in the production of sugar catabolites, bacteriocins, exopolysaccharides, and aromas, in probiotic and biosafety characteristics, and in the stress response. Also, genes linked to negative traits, such as antibiotic resistance and virulence, are represented. As LAB ecosystems contain a variety of species, there was a more global focus on these specific functional properties. Thus, an algorithm was used to design gene-specific oligonucleotides that preferably hybridize with multiple LAB species, thereby allowing controlled cross-hybridization. For proof of concept, the microarray composed of 2,269 30-mer oligonucleotides focused on LAB species that are prevalent in sourdough ecosystems. Validation hybridizations using DNA and RNA from 18 LAB strains, covering 86% of all the oligonucleotides, showed that there were wide ranges in intensity and high reproducibility between microarrays.

FIG. 1.

Reproducibility of the background-corrected intensities (Fg − Bg, where Fg is the foreground intensity and Bg is the background intensity) for two hybridizations using DNA of each of the four LAB species with the highest number of oligonucleotides on the microarray: Lactobacillus plantarum LMG 9211, Lactobacillus acidophilus IBB 801, Enterococcus faecalis LMG 8222, and Lactococcus lactis subsp. lactis LDV 22186. The black symbols represent spots for species-specific and cross-hybridizing oligonucleotides. The gray symbols represent spots for unrelated oligonucleotides.