Resveratrol Favors Adhesion and Biofilm Formation of Lacticaseibacillus paracasei subsp. paracasei Strain ATCC334.

Bacterial strains of the Lactobacillaceae family are widely used as probiotics for their multifaceted potential beneficial properties. However, no official recommendations for their clinical use exist since, in many cases, oral administrations of these bacteria displayed limited beneficial effects in human. Additional research is thus needed to improve the efficiency of existing strains with strong potential. In this context, we assess in vitro the effects of nine polyphenols to stimulate biofilm formation by lactobacilli, a feature enhancing their functionalities. Among these polyphenols, we identify trans-Resveratrol (referred to hereafter as Resveratrol) as a potent inducer of biofilm formation by Lacticaseibacillus paracasei (formerly designated as Lactobacillus paracasei) ATCC334 strain. This effect is strain-dependent and relies on the enhancement of L. paracasei adhesion to abiotic and biotic surfaces, including intestinal epithelial cells. Mechanistically, Resveratrol modify physico-chemical properties of the bacterial surface and thereby enhances L. paracasei aggregation, subsequently facilitating adhesion and biofilm development. Together, our in vitro data demonstrate that Resveratrol might be used to modulate the behavior of Lactobacilli with probiotic properties. Combination of probiotics and polyphenols could be considered to enhance the probiotic functionalities in further in vivo studies.

Biofilms of Lactobacillus plantarum and Lactobacillus fermentum: Effect on stress responses, antagonistic effects on pathogen growth and immunomodulatory properties.

Few studies have extensively investigated probiotic functions associated with biofilms. Here, we show that strains of Lactobacillus plantarum and Lactobacillus fermentum are able to grow as biofilm on abiotic surfaces, but the biomass density differs between strains. We performed microtiter plate biofilm assays under growth conditions mimicking to the gastrointestinal environment. Osmolarity and low concentrations of bile significantly enhanced Lactobacillus spatial organization. Two L. plantarum strains were able to form biofilms under high concentrations of bile and mucus. We used the agar well-diffusion method to show that supernatants from all Lactobacillus except the NA4 isolate produced food pathogen inhibitory molecules in biofilm. Moreover, TNF-α production by LPS-activated human monocytoid cells was suppressed by supernatants from Lactobacillus cultivated as biofilms but not by planktonic culture supernatants. However, only L. fermentum NA4 showed anti-inflammatory effects in zebrafish embryos fed with probiotic bacteria, as assessed by cytokine transcript level (TNF-α, IL-1ß and IL-10). We conclude that the biofilm mode of life is associated with beneficial probiotic properties of lactobacilli, in a strain dependent manner. Those results suggest that characterization of isolate phenotype in the biofilm state could be additional valuable information for the selection of probiotic strains.

The biofilm mode of life boosts the anti-inflammatory properties of Lactobacillus.

The predominant form of life for microorganisms in their natural habitats is the biofilm mode of growth. The adherence and colonization of probiotic bacteria are considered as essential factors for their immunoregulatory function in the host. Here, we show that Lactobacillus casei ATCC334 adheres to and colonizes the gut of zebrafish larvae. The abundance of pro-inflammatory cytokines and the recruitment of macrophages were low when inflammation was induced in probiotic-fed animals, suggesting that these bacteria have anti-inflammatory properties. We treated human macrophage-differentiated monocytic THP-1 cells with supernatants of L. casei ATCC334 grown in either biofilm or planktonic cultures. TNF-α production was suppressed and the NF-κB pathway was inhibited only in the presence of supernatants from biofilms. We identified GroEL as the biofilm supernatant compound responsible, at least partially, for this anti-inflammatory effect. Gradual immunodepletion of GroEL demonstrated that the abundance of GroEL and TNF-α were inversely correlated. We confirmed that biofilm development in other Lactobacillus species affects the immune response. The biofilms supernatants of these species also contained large amounts of GroEL. Thus, our results demonstrate that the biofilm enhances the immunomodulatory effects of Lactobacillus sp. and that secreted GroEL is involved in this beneficial effect.

Long-term intake of Lactobacillus helveticus enhances bioavailability of omega-3 fatty acids in the mouse retina.

Omega-3 (n-3) polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA), are required for the structure and function of the retina. Several observational studies indicate that consumption of a diet with relatively high levels of n-3 PUFAs, such as those provided by fish oils, has a protective effect against the development of age-related macular degeneration. Given the accumulating evidence showing the role of gut microbiota in regulating retinal physiology and host lipid metabolism, we evaluated the potential of long-term dietary supplementation with the Gram-positive bacterium Lactobacillus helveticus strain VEL12193 to modulate the retinal n-3 PUFA content. A set of complementary approaches was used to study the impact of such a supplementation on the gut microbiota and host lipid/fatty acid (FA) metabolism. L. helveticus-supplementation was associated with a decrease in retinal saturated FAs (SFAs) and monounsaturated FAs (MUFAs) as well as an increase in retinal n-3 and omega-6 (n-6) PUFAs. Interestingly, supplementation with L. helveticus enriched the retina in C22:5n-3 (docosapentaenoic acid, DPA), C22:6n-3 (DHA), C18:2n-6 (linoleic acid, LA) and C20:3n-6 (dihomo gamma-linolenic acid, DGLA). Long-term consumption of L. helveticus also modulated gut microbiota composition and some changes in OTUs abundance correlated with the retinal FA content. This study provides a proof of concept that targeting the gut microbiota could be an effective strategy to modulate the retinal FA content, including that of protective n-3 PUFAs, thus opening paths for the design of novel preventive and/or therapeutical strategies for retinopathies.

The oligomer plasticity of the small heat-shock protein Lo18 from Oenococcus oeni influences its role in both membrane stabilization and protein protection.

The ability of the small Hsp (heat-shock protein) Lo18 from Oenococcus oeni to modulate the membrane fluidity of liposomes or to reduce the thermal aggregation of proteins was studied as a function of the pH in the range 5-9. We have determined by size-exclusion chromatography and analytical ultracentrifugation that Lo18 assembles essentially as a 16-mer at acidic pH. Its quaternary structure evolves to a mixture of lower molecular mass oligomers probably in dynamic equilibrium when the pH increases. The best Lo18 activities are observed at pH 7 when the particle distribution contains a major proportion of dodecamers. At basic pH, particles corresponding to a dimer prevail and are thought to be the building blocks leading to oligomerization of Lo18. At acidic pH, the dimers are organized in a double-ring of stacked octamers to form the 16-mer as shown by the low-resolution structure determined by electron microscopy. Experiments performed with a modified protein (A123S) shown to preferentially form dimers confirm these results. The α-crystallin domain of Methanococcus jannaschii Hsp16.5, taken as a model of the Lo18 counterpart, fits with the electron microscopy envelope of Lo18.

Membrane vesicles released by Lacticaseibacillus casei BL23 inhibit the biofilm formation of Salmonella Enteritidis.

Biofilms represent a major concern in the food industry and healthcare. The use of probiotic bacteria and their derivatives as an alternative to conventional treatments to fight biofilm development is a promising option that has provided convincing results in the last decades. Recently, membrane vesicles (MVs) produced by probiotics have generated considerable interest due to the diversity of roles they have been associated with. However, the antimicrobial activity of probiotic MVs remains to be studied. In this work, we showed that membrane vesicles produced by Lacticaseibacillus casei BL23 (LC-MVs) exhibited strong antibiofilm activity against Salmonella enterica serovar Enteritidis (S. Enteritidis) without affecting bacterial growth. Furthermore, we found that LC-MVs affected the early stages of S. Enteritidis biofilm development and prevented attachment of bacteria to polystyrene surfaces. Importantly, LC-MVs did not impact the biomass of already established biofilms. We also demonstrated that the antibiofilm activity depended on the proteins associated with the LC-MV fraction. Finally, two peptidoglycan hydrolases (PGHs) were found to be associated with the antibiofilm activity of LC-MVs. Overall, this work allowed to identify the antibiofilm properties of LC-MVs and paved the way for the use of probiotic MVs against the development of negative biofilms.

Significant influence of four highly conserved amino-acids in lipochaperon-active sHsps on the structure and functions of the Lo18 protein.

To cope with environmental stresses, bacteria have developed different strategies, including the production of small heat shock proteins (sHSP). All sHSPs are described for their role as molecular chaperones. Some of them, like the Lo18 protein synthesized by Oenococcus oeni, also have the particularity of acting as a lipochaperon to maintain membrane fluidity in its optimal state following cellular stresses. Lipochaperon activity is poorly characterized and very little information is available on the domains or amino-acids key to this activity. The aim in this paper is to investigate the importance at the protein structure and function level of four highly conserved residues in sHSP exhibiting lipochaperon activity. Thus, by combining in silico, in vitro and in vivo approaches the importance of three amino-acids present in the core of the protein was shown to maintain both the structure of Lo18 and its functions.

Tyrosine-containing peptides are precursors of tyramine produced by Lactobacillus plantarum strain IR BL0076 isolated from wine.

BACKGROUND: Biogenic amines are molecules with allergenic properties. They are found in fermented products and are synthesized by lactic acid bacteria through the decarboxylation of amino acids present in the food matrix. The concentration of biogenic amines in fermented foodstuffs is influenced by many environmental factors, and in particular, biogenic amine accumulation depends on the quantity of available precursors. Enological practices which lead to an enrichment in nitrogen compounds therefore favor biogenic amine production in wine. Free amino acids are the only known precursors for the synthesis of biogenic amines, and no direct link has previously been demonstrated between the use of peptides by lactic acid bacteria and biogenic amine synthesis. RESULTS: Here we demonstrate for the first time that a Lactobacillus plantarum strain isolated from a red wine can produce the biogenic amine tyramine from peptides containing tyrosine. In our conditions, most of the tyramine was produced during the late exponential growth phase, coinciding with the expression of the tyrDC and tyrP genes. The DNA sequences of tyrDC and tyrP in this strain share 98% identity with those in Lactobacillus brevis consistent with horizontal gene transfer from L. brevis to L. plantarum. CONCLUSION: Peptides amino acids are precursors of biogenic amines for Lactobacillus plantarum strain IR BL0076.

Spontaneous Prophage Induction Contributes to the Production of Membrane Vesicles by the Gram-Positive Bacterium Lacticaseibacillus casei BL23.

The formation of membrane vesicles (MVs) by Gram-positive bacteria has gained increasing attention over the last decade. Recently, models of vesicle formation have been proposed and involve the digestion of the cell wall by prophage-encoded or stress-induced peptidoglycan (PG) hydrolases and the inhibition of PG synthesis by ß-lactam antibiotics. The impact of these mechanisms on vesicle formation is largely dependent on the strain and growth conditions. To date, no information on the production of vesicles by the lactobacilli family has been reported. Here, we aimed to characterize the MVs released by the Gram-positive bacteria Lacticaseibacillus casei BL23 and also investigated the mechanisms involved in vesicle formation. Using electron microscopy, we established that the size of the majority of L. casei BL23 vesicles ranged from 50 to 100 nm. Furthermore, we showed that the vesicles were released consistently throughout the growth of the bacteria in standard culture conditions. The protein composition of the vesicles released in the supernatant was identified and a significant number of prophage proteins was detected. Moreover, using a mutant strain harboring a defective PLE2 prophage, we were able to show that the spontaneous and mitomycin-triggered induction of the prophage PLE2 contribute to the production of MVs by L. casei BL23. Finally, we also demonstrated the influence of prophages on the membrane integrity of bacteria. Overall, our results suggest a key role of the prophage PLE2 in the production of MVs by L. casei BL23 in the absence or presence of genotoxic stress. IMPORTANCE The last few decades have demonstrated that membrane vesicles (MVs) produced by microorganisms can have a wide variety of functions. This diversity places MVs at the crossroads of major research topics in current microbiology such as antibiotic resistance, horizontal gene transfer, cell communication, biofilm development, bacteriophage resistance, and pathogenesis. In particular, vesicles produced by probiotic strains have been shown to play a significant role in their beneficial effects. Thus, the study of vesicle biogenesis is a key element for promoting and improving their release. Overall, our results suggest a key role of spontaneous and mitomycin-triggered prophage induction in MV production by the Gram-positive bacteria Lacticaseibacillus casei BL23. This phenomenon is of great interest as prophage-induced MVs could potentially influence bacterial behavior, stress resistance, and vesicle functions.

Lactobacillus stress protein GroEL prevents colonic inflammation.

BACKGROUND: We previously showed that supernatants of Lactobacillus biofilms induced an anti-inflammatory response by affecting the secretion of macrophage-derived cytokines, which was abrogated upon immunodepletion of the stress protein GroEL. METHODS: We purified GroEL from L. reuteri and analysed its anti-inflammatory properties in vitro in human macrophages isolated from buffy coats, ex vivo in explants from human biopsies and in vivo in a mouse model of DSS induced intestinal inflammation. As a control, we used GroEL purified (LPS-free) from E. coli. RESULTS: We found that L. reuteri GroEL (but not E. coli GroEL) inhibited pro-inflammatory M1-like macrophages markers, and favored M2-like markers. Consequently, L. reuteri GroEL inhibited pro-inflammatory cytokines (TNFα, IL-1ß, IFNγ) while favouring an anti-inflammatory secretome. In colon tissues from human biopsies, L. reuteri GroEL was also able to decrease markers of inflammation and apoptosis (caspase 3) induced by LPS. In mice, we found that rectal administration of L. reuteri GroEL (but not E. coli GroEL) inhibited all signs of haemorrhagic colitis induced by DSS including intestinal mucosa degradation, rectal bleeding and weight loss. It also decreased intestinal production of inflammatory cytokines (such as IFNγ) while increasing anti-inflammatory IL-10 and IL-13. These effects were suppressed when animals were immunodepleted in macrophages. From a mechanistic point of view, the effect of L. reuteri GroEL seemed to involve TLR4, since it was lost in TRL4-/- mice, and the activation of a non-canonical TLR4 pathway. CONCLUSIONS: L. reuteri GroEL, by affecting macrophage inflammatory features, deserves to be explored as an alternative to probiotics.

Characterization of the CtsR stress response regulon in Lactobacillus plantarum.

Lactobacillus plantarum ctsR was characterized. ctsR was found to be cotranscribed with clpC and induced in response to various abiotic stresses. ctsR deletion conferred a heat-sensitive phenotype with peculiar cell morphological features. The transcriptional pattern of putative CtsR regulon genes was examined in the Delta ctsR mutant. Direct CtsR-dependent regulation was demonstrated by DNA-binding assays using recombinant CtsR and the promoters of the ctsR-clpC operon and hsp1.

Intestinal release of biofilm-like microcolonies encased in calcium-pectinate beads increases probiotic properties of Lacticaseibacillus paracasei.

In this study, we show that calcium pectinate beads (CPB) allow the formation of 20 µm spherical microcolonies of the probiotic bacteria Lacticaseibacillus paracasei (formerly designated as Lactobacillus paracasei) ATCC334 with a high cell density, reaching more than 10 log (CFU/g). The bacteria within these microcolonies are well structured and adhere to a three-dimensional network made of calcium-pectinate through the synthesis of extracellular polymeric substances (EPS) and thus display a biofilm-like phenotype, an attractive property for their use as probiotics. During bacterial development in the CPB, a coalescence phenomenon arises between neighboring microcolonies accompanied by their peripheral spatialization within the bead. Moreover, the cells of L. paracasei ATCC334 encased in these pectinate beads exhibit increased resistance to acidic stress (pH 1.5), osmotic stress (4.5 M NaCl), the freeze-drying process and combined stresses, simulating the harsh conditions encountered in the gastrointestinal (GI) tract. In vivo, the oral administration of CPB-formulated L. paracasei ATCC334 in mice demonstrated that biofilm-like microcolonies are successfully released from the CPB matrix in the colonic environment. In addition, these CPB-formulated probiotic bacteria display the ability to reduce the severity of a DSS-induced colitis mouse model, with a decrease in colonic mucosal injuries, less inflammation, and reduced weight loss compared to DSS control mice. To conclude, this work paves the way for a new form of probiotic administration in the form of biofilm-like microcolonies with enhanced functionalities.

Inhibition of mitophagy drives macrophage activation and antibacterial defense during sepsis.

Mitochondria have emerged as key actors of innate and adaptive immunity. Mitophagy has a pivotal role in cell homeostasis, but its contribution to macrophage functions and host defense remains to be delineated. Here, we showed that lipopolysaccharide (LPS) in combination with IFN-γ inhibited PINK1-dependent mitophagy in macrophages through a STAT1-dependent activation of the inflammatory caspases 1 and 11. In addition, we demonstrated that the inhibition of mitophagy triggered classical macrophage activation in a mitochondrial ROS-dependent manner. In a murine model of polymicrobial infection (cecal ligature and puncture), adoptive transfer of Pink1-deficient bone marrow or pharmacological inhibition of mitophagy promoted macrophage activation, which favored bactericidal clearance and led to a better survival rate. Reciprocally, mitochondrial uncouplers that promote mitophagy reversed LPS/IFN-γ-mediated activation of macrophages and led to immunoparalysis with impaired bacterial clearance and lowered survival. In critically ill patients, we showed that mitophagy was inhibited in blood monocytes of patients with sepsis as compared with nonseptic patients. Overall, this work demonstrates that the inhibition of mitophagy is a physiological mechanism that contributes to the activation of myeloid cells and improves the outcome of sepsis.

The Phenotypic Analysis of Lactobacillus plantarum shsp Mutants Reveals a Potential Role for hsp1 in Cryotolerance.

Small heat shock proteins (sHSPs) are ubiquitous, low molecular weight (MW) proteins that share a conserved alpha-crystallin domain. sHSPs oligomers exhibit chaperon-like activities by interacting with unfolded substrates, thereby preventing their aggregation and precipitation. Unlike most lactobacilli, which have single shsp genes, three different sHSP-encoding genes, i.e., hsp1, hsp2, and hsp3, were previously identified in the probiotic Lactobacillus plantarum WCFS1. Early studies, including the characterization of the knock out (KO) mutant for hsp2, indicated a different organization and transcriptional regulation of these genes and suggested that the three L. plantarum sHSPs might accomplish different tasks in stress response. To unravel the role of sHSPs, KO mutants of hsp1 and hsp3 were generated using a Cre-lox based system. Mutation of either genes resulted in impaired growth capacity under normal conditions, heat-stress and stresses typically found during host interactions and food technological process. However, survival to heat shock and the level of thermal stabilization of cytoplasmic proteins were similar between mutants and parental strain. Transcriptional analysis revealed that in the mutant genetic backgrounds there is an upregulated basal expression of the un-mutated mate hsps and other stress-related genes, which may compensate for the loss of HSP function, hence possibly accounting for the lack of a remarkable susceptibility to heat challenge. HSP3 seemed relevant for the induction of thermotolerance, while HSP1 was required for improved cryotolerance. Cell surface properties and plasma membrane fluidity were investigated to ascertain the possible membrane association of sHSP. Intriguingly, the loss of hsp1 was associated to a lower level of maximal membrane fluidity upon heat stress. A role for HSP1 in controlling and improving membrane fluidity is suggested which may pertains its cryoprotective function.

Disentangling the effect of host genetics and gut microbiota on resistance to an intestinal parasite.

Resistance to infection is a multifactorial trait, and recent work has suggested that the gut microbiota can also contribute to resistance. Here, we performed a fecal microbiota transplant to disentangle the contribution of the gut microbiota and host genetics as drivers of resistance to the intestinal nematode Heligmosomoides polygyrus. We transplanted the microbiota of a strain of mice (SJL), resistant to H. polygyrus, into a susceptible strain (CBA) and vice-versa. We predicted that if the microbiota shapes resistance to H. polygyrus, the FMT should reverse the pattern of resistance between the two host strains. The two host strains had different microbiota diversities and compositions before the start of the experiment, and the FMT altered the microbiota of recipient mice. One mouse strain (SJL) was more resistant to colonization by the heterologous microbiota, and it maintained its resistance profile to H. polygyrus (lower parasite burden) independently of the FMT. On the contrary, CBA mice harbored parasites with lower fecundity during the early stage of the infection, and had an up-regulated expression of the cytokine IL-4 (a marker of H. polygyrus resistance) after receiving the heterologous microbiota. Therefore, while host genetics remains the main factor shaping the pattern of resistance to H. polygyrus, the composition of the gut microbiota also seems to play a strain-specific role.

Listeria monocytogenes EGD-e biofilms: no mushrooms but a network of knitted chains.

Listeria monocytogenes is a food pathogen that can attach on most of the surfaces encountered in the food industry. Biofilms are three-dimensional microbial structures that facilitate the persistence of pathogens on surfaces, their resistance toward antimicrobials, and the final contamination of processed goods. So far, little is known about the structural dynamics of L. monocytogenes biofilm formation and its regulation. The aims of this study were, by combining genetics and time-lapse laser-scanning confocal microscopy (LSCM), (i) to characterize the structural dynamics of L. monocytogenes EGD-e sessile growth in two nutritional environments (with or without a nutrient flow), and (ii) to evaluate the possible role of the L. monocytogenes agr system during biofilm formation by tracking the spatiotemporal fluorescence expression of a green fluorescent protein (GFP) reporter system. In the absence of nutrient flow (static conditions), unstructured biofilms composed of a few layers of cells that covered the substratum were observed. In contrast, when grown under dynamic conditions, L. monocytogenes EGD-e biofilms were highly organized. Indeed, ball-shaped microcolonies were surrounded by a network of knitted chains. The spatiotemporal tracking of fluorescence emitted by the GFP reporter system revealed that agr expression was barely detectable under static conditions, but it progressively increased during 40 h under dynamic conditions. Moreover, spatial analysis revealed that agr was expressed preferentially in cells located outside the microcolonies. Finally, the in-frame deletion of agrA, which encodes a transcriptional regulator, resulted in a decrease in initial adherence without affecting the subsequent biofilm development.

Interactions in dual species biofilms between Listeria monocytogenes EGD-e and several strains of Staphylococcus aureus.

Six environmental isolates of Staphylococcus aureus and one collection strain were investigated for their ability to form monospecies biofilms and dual species biofilms with Listeria monocytogenes EGD-e on stainless steel coupons. All isolates were able to grow as biofilms but their ability to form monospecies biofilms differed. The population of L. monocytogenes EGD-e in dual species biofilms was not affected by the presence of S. aureus isolates except for strain CIP 53.156. The effect of L. monocytogenes EGD-e on the population of S. aureus was strain dependent: S. aureus population either increased or decreased or was not affected in the presence of L. monocytogenes EGD-e in dual species biofilms. Dual species biofilms were grown with L. monocytogenes EGD-e and the strain CIP 53.156 of S. aureus on stainless steel coupons under batch and dynamic conditions. Higher sessile populations of L. monocytogenes EGD-e were observed in the presence of S. aureus CIP 53.156. Microscope observations by scanning electron microscopy (SEM) revealed an intimate association of L. monocytogenes EGD-e and S. aureus CIP 53.156 in dual species biofilms. An increase of the number of L. monocytogenes EGD-e cells was observed in the presence of S. aureus CIP 53.156 cell-free supernatant. This activity was retained after ultrafiltation (<3 kDa), was heat stable but was lost after proteinase K treatment.

Resveratrol-Induced Xenophagy Promotes Intracellular Bacteria Clearance in Intestinal Epithelial Cells and Macrophages.

Autophagy is a lysosomal degradation process that contributes to host immunity by eliminating invasive pathogens and the modulating inflammatory response. Several infectious and immune disorders are associated with autophagy defects, suggesting that stimulation of autophagy in these diseases should be beneficial. Here, we show that resveratrol is able to boost xenophagy, a selective form of autophagy that target invasive bacteria. We demonstrated that resveratrol promotes in vitro autophagy-dependent clearance of intracellular bacteria in intestinal epithelial cells and macrophages. These results were validated in vivo using infection in a transgenic GFP-LC3 zebrafish model. We also compared the ability of resveratrol derivatives, designed to improve the bioavailability of the parent molecule, to stimulate autophagy and to induce intracellular bacteria clearance. Together, our data demonstrate the ability of resveratrol to stimulate xenophagy, and thereby enhance the clearance of two invasive bacteria involved life-threatening diseases, Salmonella Typhimurium and Crohn's disease-associated Adherent-Invasive Escherichia coli. These findings encourage the further development of pro-autophagic nutrients to strengthen intestinal homeostasis in basal and infectious states.

Production of the small heat shock protein Lo18 from Oenococcus oeni in Lactococcus lactis improves its stress tolerance.

Lactococcus lactis is a lactic acid bacterium widely used in cheese and fermented milk production. During fermentation, L. lactis is subjected to acid stress that impairs its growth. The small heat shock protein (sHsp) Lo18 from the acidophilic species Oenococcus oeni was expressed in L. lactis. This sHsp is known to play an important role in protein protection and membrane stabilization in O. oeni. The role of this sHsp could be studied in L. lactis, since no gene encoding for sHsp has been detected in this species. L. lactis subsp. cremoris strain MG1363 was transformed with the pDLhsp18 plasmid, which is derived from pDL278 and contains the hsp18 gene (encoding Lo18) and its own promoter sequence. The production of Lo18 during stress conditions was checked by immunoblotting and the cellular distribution of Lo18 in L. lactis cells after heat shock was determined. Our results clearly indicated a role for Lo18 in cytoplasmic protein protection and membrane stabilization during stress. The production of sHsp in L. lactis improved tolerance to heat and acid conditions in this species. Finally, the improvement of the L. lactis survival in milk medium thanks to Lo18 was highlighted, suggesting an interesting role of this sHsp. These findings suggest that the expression of a sHsp by a L. lactis strain results in greater resistance to stress, and, can consequently enhance the performances of industrial strains.

Inactivation of the ftsH gene of Lactobacillus plantarum WCFS1: effects on growth, stress tolerance, cell surface properties and biofilm formation.

FtsH proteins are ubiquitous membrane-bound, ATP-dependent metalloproteases of the AAA family. In eubacteria, FtsH is involved in protein quality control under stress conditions. Lactobacillus plantarum is a widespread lactic acid bacterium that is encountered in several fermented food, including dairy products, vegetables and meat. In the present work the expression of the ftsH gene of L. plantarum was studied by quantitative real time RT-PCR in bacterial cultures subjected to various abiotic stresses. Both oxidative stress and addition of a membrane-fluidizing agent induced ftsH transcription, while a depletion of carbon-source repressed its mRNA level. Mutants deprived of the FtsH protease exhibited remarkable sensitivity to elevated temperature and increased salt concentration; conversely, overexpression of ftsH resulted in increased thermotolerance and resistance to salt. FtsH mutant had a reduced capacity to form biofilms on abiotic surfaces and exhibited different cell surface physico-chemical properties with respect to the wild type strain.