Dynamic Analysis of the Lactococcus lactis Transcriptome in Cheeses Made from Milk Concentrated by Ultrafiltration Reveals Multiple Strategies of Adaptation to Stresses

Abstract

Lactococcus lactis is used extensively for the production of various cheeses. At every stage of cheese fabrication, L. lactis has to face several stress-generating conditions that result from its own modification of the environment as well as externally imposed conditions. We present here the first in situ global gene expression profile of L. lactis in cheeses made from milk concentrated by ultrafiltration (UF-cheeses), a key economical cheese model. The transcriptomic response of L. lactis was analyzed directly in a cheese matrix, starting from as early as 2 h and continuing for 7 days. The growth of L. lactis stopped after 24 h, but metabolic activity was maintained for 7 days. Conservation of its viability relied on an efficient proteolytic activity measured by an increasing, quantified number of free amino acids in the absence of cell lysis. Extensive downregulation of genes under CodY repression was found at day 7. L. lactis developed multiple strategies of adaptation to stressful modifications of the cheese matrix. In particular, expression of genes involved in acidic-and oxidativestress responses was induced. L. lactis underwent unexpected carbon limitation characterized by an upregulation of genes involved in carbon starvation, principally due to the release of the CcpA control. We report for the first time that in spite of only moderately stressful conditions, lactococci phage is repressed under UF-cheese conditions. Lactic acid bacteria, particularly Lactococcus lactis, have a long history of use in milk fermentation, from small-scale traditional operations to well-controlled industrial applications. Recent developments of molecular tools have unraveled the genetics, physiology, and metabolism of this economically very important microorganism. However, interpretation has always been limited by the lack of knowledge regarding in situ bacterial physiology. Technological properties (e.g., acidification, proteolytic or lipolytic activity, and bacteriocin production) can easily be shown and quantified in vitro but have hardly ever been verified in a complex solid matrix, due to local intrinsic factors. There have been several successful attempts to measure the DNA and rRNA extracted directly from a solid food (or environmental) matrix in order to estimate either the predominant species and/or the overall level of metabolic activity of the species (11,33). In a few cases, the expression of genes of technological interest from extracted mRNAs has been checked (42). The global gene or protein expression of L. lactis has been characterized in milk (15, 34) but not directly in cheese, the corresponding solid dairy matrix.Over the last few years, functional-genomics approaches, including transcriptomics, have been increasingly used to obtain global gene expression profiles, thereby providing a comprehensive view of microorganism physiology. So far, such global approaches in food microbiology and in situ have been poorly documented. Recently, Bachmann et al. presented the genetic responses of L. lactis in mixed cu...

Inhibition of Staphylococcus aureus Invasion into Bovine Mammary Epithelial Cells by Contact with Live Lactobacillus casei

“…casei inhibition of S. aureus internalization probably involves one or more means acting alone or in combination: (i) modulation of bMEC physiology or integrity induced by contact; (ii) direct effect on S. aureus, including coaggregation, as observed for vaginal lactobacilli (47,48), although preliminary experiments indicated that L. casei 667 exhibited poor aggregative abilities (data not shown); (iii) inhibition of S. aureus virulence expression, including major virulence regulators, as previously reported (48)(49)(50)(51); and (iv) competition for attachment sites involved in internalization. In agreement with a competition mechanism, the L. casei internalization rate was also affected by S. aureus.…”

Cloning, Expression, and Functional Characterization of Secondary Amino Acid Transporters of Lactococcus lactis

“…During fermentation in milk, some amino acids are excreted by L. lactis (43)(44)(45), indicating that the amino acid composition of the peptides taken up after the hydrolysis of casein by PrtP is not matching the requirements for biosynthesis. The excretion of excess amino acids was proposed to be coupled to the excretion of a proton (proton symport), which would contribute to the generation of metabolic energy in the form of proton motive force (PMF) (energy-recycling model).…”

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products

“…cremoris strains under conditions simulating cheddar cheese manufacture (Taïbi et al, 2011) and for the study of iron acquisition by Arthrobacter arilaitensis in experimental cheeses (Monnet et al, 2012 Experimental cheeses L. lactis subsp. lactis bcaT, codY, serA, cysK, gltD, lacC, gapA, gapB, pdhB, aldB, butA, noxE, murF, dnaK, chiA, pepN, gyrB, pi139, pi302 Experimental cheeses (Cretenet et al, 2011) L. lactis subsp. cremoris bcaT,clpE,dnaG,gapA,glyA,groEL,oppA,pepQ,purD,ldh,holin1,holin2 Experimental cheeses (Taïbi et al, 2011) Lb.…”

“…The calculated fold-change does not reflect the specific gene expression of each population, but rather an expression taking into account the total amount of 16S rRNA. Cretenet et al (2011) quantified the expression of several genes from L. lactis in model cheeses made from ultrafiltered milk, using gyrB (DNA gyrase subunit B) as reference gene. The histidine decarboxylase gene (hdcA) present in certain Str.…”

“…RNA extraction methods involving prior separation of the cells from cheeses and other dairy products have been used in several studies (Randazzo et al, 2002;Bleve et al, 2003;Sanchez et al, 2006;Bogovic Matijasic et al, 2007;Smeianov et al, 2007;Makhzami et al, 2008;Rantsiou et al, 2008a;Rantsiou et al, 2008b;Ulvé et al, 2008;Duquenne et al, 2010;Falentin et al, 2010;Cretenet et al, 2011;La Gioia et al, 2011;Masoud et al, 2011;Taïbi et al, 2011). The recovery of microbial cells is done following similar protocols than for DNA extraction methods (see above).…”