cDNA-amplified fragment length polymorphism to study the transcriptional responses of Lactobacillus rhamnosus growing in cheese-like medium

Abstract

Aims: Lactobacillus rhamnosus is a dominant species during Parmigiano Reggiano cheese ripening and exhibits a great adaptability to unfavourable growth conditions. Gene expression of a Lact. rhamnosus, isolated from Parmigiano Reggiano cheese, grown in a rich medium (MRS) and in a cheese‐like medium (CB) has been compared by a novel cDNA‐amplified fragment length polymorphism (cDNA‐AFLP) protocol. Methods and Results: Two techniques, capillary and gel electrophoresis cDNA‐AFLP, were applied to generate unique transcript tags from reverse‐transcribed messenger RNA using the immobilization of biotinylated 3′‐terminal cDNA fragments on streptavidin‐coated Dynabeads. The use of three pairs of primers allowed detecting 64 genes expressed in MRS and 96 in CB. Different transcripts were observed when Lact. rhamnosus was cultured on CB and MRS. Conclusions: The cDNA‐AFLP approach proved to be able to show that Lact. rhamnosus modifies the expression of a large part of genes when cultivated in CB compared with growth under optimal conditions (MRS). In particular, the profiles of the strain grown in CB were more complex probably because the cells activate different metabolic pathways to generate energy and to respond to the environmental changes. Significance and Impact of Study: This is the first research on Lact. rhamnosus isolated from cheese and represents one of the few concerning bacterial transcriptomic analysis towards cDNA‐AFLP approaches.

Transcriptomic clues to understand the growth of Lactobacillus rhamnosus in cheese

“…The strain was cultivated in MRS broth (Oxoid) or Cheese Broth (CB) at 30°C, under anaerobiosis, for 24 or 48 h, respectively. CB, a culture medium that mimics raw-milk long-ripened cheese, was prepared according to the modified protocol described by Bove et al [16,18]. …”

“…All RNAs were of sufficient quantity (>350 ng/μl) and high quality (A260/A280 ratio 2.0 to 2.1). After a step of mRNA enrichment and polyadenylation of RNA transcripts, cDNA was synthesized by reverse transcription (RT) using a biotinylated oligo (dT), following the protocol reported by Bove et al [18]. For qPCR, single-stranded cDNA was prepared in a final volume of 20 μl from total RNA using an oligo (dT) primer and SuperScript™ II reverse transcriptase (Invitrogen), according to the manufacturer’s instructions.…”

“…cDNA-AFLP analysis was carried out as described by Bove et al [18]. The protocol is based on the production of cDNA-AFLP fragments that are detected using infrared dye (IRD) detection technology and the Odyssey Infrared Imaging System.…”

“…Differently from MRS, which is the standard laboratory medium for lactobacilli [17] and is considered to be a rich substrate with glucose as the primary carbon source for microbial growth, CB is an experimental medium formulated with 20-month-ripened PR cheese [10,16,18], that tries to mimic the nutritional composition of PR cheese during ripening. PR cheese, and thus CB, is considered to be a substrate poor in carbohydrates and characterized by the absence of milk sugar, lactose.…”

Evolution of microbiological analytical methods for dairy industry needs

“…Polymerase chain reaction techniques were also used to evaluate physiological states and viability of microorganisms during products processes (Lahtinen et al, 2008; Marco and Kleerebezem, 2008; García-Cayuela et al, 2009; Randazzo et al, 2009; Matijašić et al, 2010; Meng et al, 2010; Bove et al, 2011). One way to distinguish between viable and dead bacteria is to use RT-qPCR which targets RNA instead of DNA (Matsuda et al, 2009; Falentin et al, 2010; Reimann et al, 2010).…”