Cloning and expression of genes involved in conidiation and surface properties of Penicillium camemberti grown in liquid and solid cultures.

International audience; Based on bioinformatic data on model fungi, the rodA and wetA genes encoding, respectively, a RodA hydrophobin protein and the WetA protein involved in conidiation mechanisms, were PCR-cloned and characterized for the first time in Penicillium camemberti. These results, completed by a sequence of the brlA gene (available in GenBank), which encodes a major transcriptional regulator also involved in the conidiation mechanism, were used to compare, by qRT-PCR, the expression of the three genes in liquid and solid cultures in a synthetic medium. While expression of the brlA and wetA genes increased dramatically in both culture conditions after 4 days of growth, expressio…

Truncated internalin A and asymptomatic Listeria monocytogenes carriage: in vivo investigation by allelic exchange

ABSTRACT Allelic exchange of the region coding for the C terminus of InlA between one epidemic (with an 80-kDa InlA) and one asymptomatic (with a 47-kDa InlA) carriage Listeria monocytogenes strain confirmed the need for this region for internalin entry in vitro. Interestingly, restoration of internalin A functionality did not result in full virulence in chicken embryo assays.

Investigation of root-associated biofilms of the human pathogenic bacterium Listeria monocytogenes

EAMERSCT1EJ2; International audience; No abstract

Listeria monocytogenes, a down-to-earth pathogen

International audience; Listeria monocytogenes is the causative agent of the food-borne life threatening disease listeriosis. This pathogenic bacterium received much attention in the endeavor of deciphering the cellular mechanisms that underlie the onset of infection and its ability to adapt to the food processing environment. Although information is available on the presence of L. monocytogenes in many environmental niches including soil, water, plants, foodstuff and animals, understanding the ecology of L. monocytogenes in outdoor environments has received less attention. Soil is an environmental niche of pivotal importance in the transmission of this bacterium to plants and animals. Soil…

Le rôle du système agr chez Listeria monocytogènes

National audience; La capacité des bactéries à développer un biofilm constitue une des principales difficultés quant à la maîtrise de la contamination : ces structures en augmentent les risques et rendent notamment les micro-organismes plus résistants aux désinfectants. Des études ont pu démontrer le rôle du système de communication agr dans la formation de biofilms de L. monocytogenes, agent pathogène particulièrement recherché en industrie agro-alimentaire.

Cloning and sequencing of the gene encoding α-acetolactate decarboxylase fromLeuconostoc oenos

The alsD gene encoding alpha-acetolactate decarboxylase was isolated from a genomic library of Leuconostoc oenos, using a screening procedure developed on microtiter plates. The nucleotide sequence of alsD encodes a putative protein of 239 amino acids showing significant similarity with other bacterial alpha-acetolactate decarboxylases. Upstream from alsD lies an open reading frame (alsS) which is highly similar to bacterial genes coding for catabolic alpha-acetolactate synthases. Northern (RNA) blotting analyses indicated the presence of a 2.4-kb dicistronic transcript of alsS and alsD. This suggests that the alsS and alsD genes are organized in a single operon.

Adaptation à l'environnement : métabolisme et communication bactérienne

Influence des conditions environnementales (statique versus dynamique) sur le développement en biofilm de Listeria monocytogenes EGD-e. Developpement en biofilm et expression d'Agr de Listeria monocytogenes

National audience

Le système [i]agr[/i] de [i]Listeria monocytogenes[/i] et la croissance en biofilm : revisite des notions de communication, quorum sensing et auto-induction

Communication orale, résumé; Parmi les mécanismes cellulaires qui participent à la mise en place des biofilms, l’échange de molécules de communication apparaît nécessaire chez de nombreux microorganismes. Listeria monocytogenes est une bactérie ubiquiste responsable de pathologies sévères chez des populations à risque (immunodéprimés, femmes enceintes, personnes âgées) en cas de consommation d’aliments contaminés. Nous avons récemment démontré que le système de communication agr est impliqué au cours de la croissance sessile de cette bactérie (Rieu et al., 2007). Par ailleurs, l’expression du système agr était hétérogène pendant la croissance en biofilm (Rieu et al., 2008) et localisée à sa…

Differential expression of the agr locus of Listeria monocytogenes EGD-e

International audience

LisRK is required for optimal fitness ofListeria monocytogenesin soil

ABSTRACTListeria monocytogenes is a food-borne pathogen responsible for the disease listeriosis. It is ubiquitously found in the environment and soil is one of its natural habitats. Listeria monocytogenes is highly capable of coping with various stressful conditions. We hypothesized that stress-responsive two-component systems such as LisRK might contribute to the adaptation of L. monocytogenes to the soil environment. Indeed, investigations of the population dynamics of wild-type and mutant strains suggest an important role of LisRK for optimal fitness of L. monocytogenes in sterile soil. Results from non-sterile soil showed that the parental strain was capable of surviving longer than mut…

Purification of Leuconostoc mesenteroides citrate lyase and cloning and characterization of the citCDEFG gene cluster

ABSTRACT A citrate lyase (EC 4.1.3.6 ) was purified 25-fold from Leuconostoc mesenteroides and was shown to contain three subunits. The first 42 amino acids of the β subunit were identified, as well as an internal peptide sequence spanning some 20 amino acids into the α subunit. Using degenerated primers from these sequences, we amplified a 1.2-kb DNA fragment by PCR from Leuconostoc mesenteroides subsp. cremoris . This fragment was used as a probe for screening a Leuconostoc genomic bank to identify the structural genes. The 2.7-kb gene cluster encoding citrate lyase of L. mesenteroides is organized in three open reading frames, citD , citE , and citF , encoding, respectively, the three ci…

Mutation of the oxaloacetate decarboxylase gene of Lactococcus lactis subsp. lactis impairs the growth during citrate metabolism

 ; Aims: Citrate metabolism generates metabolic energy through the generation of a membrane potential and a pH gradient. The purpose of this work was to study the influence of oxaloacetate decarboxylase in citrate metabolism and intracellular pH maintenance in relation to acidic conditions. Methods and Results: A Lactococcus lactis oxaloacetate decarboxylase mutant [ILCitM (pFL3)] was constructed by double homologous recombination. During culture with citrate, and whatever the initial pH, the growth rate of the mutant was lower. In addition, the production of diacetyl and acetoin was altered in the mutant strain. However, our results indicated no relationship with a change in the maintenanc…

Agr system of Listeria monocytogenes EGD-e: role in adherence and differential expression pattern.

ABSTRACT In this study, we investigated the agrBDCA operon in the pathogenic bacterium Listeria monocytogenes EGD-e. In-frame deletion of agrA and agrD resulted in an altered adherence and biofilm formation on abiotic surfaces, suggesting the involvement of the agr system of L. monocytogenes during the early stages of biofilm formation. Real-time PCR experiments indicated that the transcript levels of agrBDCA depended on the stage of biofilm development, since the levels were lower after the initial attachment period than during biofilm growth, whereas transcription during planktonic growth was not growth phase dependent. The mRNA quantification data also suggested that the agr system was a…

Regulation of stress response in Oenococcus oeni as a function of environmental changes and growth phase

International audience; Oenococcus oeni is a lactic acid bacterium which is able to grow in wine and perform malolactic fermentation. To survive and grow in such a harsh environment as wine, O. oeni uses several mechanisms of resistance including stress protein synthesis. The molecular characterisation of three stress genes hsp18, clpX, trxA encoding for a small heat shock protein, an ATPase regulation component of ClpP protease and a thioredoxin, respectively, allow us to suggest the existence in O. oeni of multiple regulation mechanisms as is the case in Bacillus subtilis. One common feature of these genes is that they are expressed under the control of housekeeping promoters. The express…

Communication and Autoinduction in the species Listeria monocytogenes

International audience; In order to withstand changes in their environment, bacteria have evolved mechanisms to sense the surrounding environment, integrate these signals and adapt their physiology to thrive under fluctuating conditions. Among these mechanisms, the ability of bacteria to exchange information between cells has become a dynamic field of interest for microbiologists over the past four decades. First described by Nelson et al.,1 this phenomenon often referred as either cell-cell communication, Quorum Sensing and/or AutoInduction involves the synthesis of small signal molecules called autoinducers. These signal molecules may be sensed by the bacterial population in the vicinity …

The Agr communication system provides a benefit to the populations of Listeria monocytogenes in soil

International audience; In this study, we investigated whether the Agr communication system of the pathogenic bacterium Listeria monocytogenes was involved in adaptation and competitiveness in soil. Alteration of the ability to communicate, either by deletion of the gene coding the response regulator AgrA (response-negative mutant) or the signal pro-peptide AgrD (signal-negative mutant), did not affect population dynamics in soil that had been sterilized but survival was altered in biotic soil suggesting that the Agr system of L. monocytogenes was involved to face the complex soil biotic environment. This was confirmed by a set of co-incubation experiments. The fitness of the response-negat…

Evidence of autoinduction heterogeneity via expression of the agr system of listeria monocytogenes at the single-cell level

ABSTRACT To investigate if the primary function of the Agr system of Listeria monocytogenes is to monitor cell density, we followed Agr expression in batch cultures, in which the autoinducer concentration was uniform, and in biofilms. Expression was heterogeneous, suggesting that the primary function of Agr is not to monitor population density.

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

ABSTRACT 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…

Investigation of rhizosphere as a niche for Listeria monocytogenes

EAUBAGROSUPBIOME; International audience; No abstract

Microbial diversity and structure are drivers of the biological barrier effect against Listeria monocytogenes in soil

International audience; Understanding the ecology of pathogenic organisms is important in order to monitor their transmission in the environment and the related health hazards. We investigated the relationship between soil microbial diversity and the barrier effect against Listeria monocytogenes invasion. By using a dilution-to-extinction approach, we analysed the consequence of eroding microbial diversity on L. monocytogenes population dynamics under standardised conditions of abiotic parameters and microbial abundance in soil microcosms. We demonstrated that highly diverse soil microbial communities act as a biological barrier against L. monocytogenes invasion and that phylogenetic compos…

Single cell analysis evidence Heterogeneous expression of the Agr communication system of Listeria monocytogenes

http://prodinra.inra.fr/record/271694EAMERSCT3 EJ3; Communication, often referred as Quorum Sensing, is involved in the adaptation of most bacteria to their environment. In the genus Listeria, the agr communication system affects the biology of Listeria monocytogenes during saprophytic life (biofilm formation) and during infection. In the present study, we investigated in situ agr expression of six isolates of L. monocytogenes by combining gfp reporters, flow cytometry and fluorescent microscopy during plantonic growth and dynamic flow cell biofilm set ups. Unexpectedly, during growth in TSB homogenised liquid cultures incubated at 25°C, while auto-inducer concentration was uniform, statist…

σB and agrA regulation in Listeria monocytogenes: Effect on survival in soil/rhizosphere under biotic and abiotic conditions

International audience; Abstract -250 words Backgrounds: Listeria monocytogenes is the agent of listeriosis, a life-threatening condition in at-risk people. Complex transmission routes between outdoor environments and the food chain result in foodstuff contamination. Sensing of environmental changes can trigger regulation of gene expression, allowing bacteria to adapt their physiology and survive. The Agr cell-cell communication system transcription regulator AgrA is triggered during several environmental conditions including soil, an important reservoir of L. monocytogenes. The RNA polymerase σB factor aids survival in several stress conditions and may be required for L. monocytogenes surv…

Survival of Listeria monocytogenes in Soil Requires AgrA-Mediated Regulation

ABSTRACT In a recent paper, we demonstrated that inactivation of the Agr system affects the patterns of survival of Listeria monocytogenes (A.-L. Vivant, D. Garmyn, L. Gal, and P. Piveteau, Front Cell Infect Microbiol 4:160, http://dx.doi.org/10.3389/fcimb.2014.00160 ). In this study, we investigated whether the Agr-mediated response is triggered during adaptation in soil, and we compared survival patterns in a set of 10 soils. The fate of the parental strain L. monocytogenes L9 (a rifampin-resistant mutant of L. monocytogenes EGD-e) and that of a Δ agrA deletion mutant were compared in a collection of 10 soil microcosms. The Δ agrA mutant displayed significantly reduced survival in these b…

Investigation of the roles of AgrA and σB regulators in Listeria monocytogenes adaptation to roots and soil

ABSTRACT Little is known about the regulatory mechanisms that ensure the survival of the food-borne bacterial pathogen Listeria monocytogenes in the telluric environment and on roots. Earlier studies have suggested a regulatory overlap between the Agr cell–cell communication system and the general stress response regulator σB. Here, we investigated the contribution of these two systems to root colonisation and survival in sterilised and biotic soil. The ability to colonise the roots of the grass Festuca arundinacea was significantly compromised in the double mutant (∆agrA∆sigB). In sterile soil at 25°C, a significant defect was observed in the double mutant, suggesting some synergy between …

Listeria monocytogenes agr system: Quorum sensing, or maybe not?

International audience

Le système Agr de Listeria monocytogenes : revisite des notions de Quorum Sensing et auto-induction par cytométrie en flux

National audience

Listeria monocytogenes Differential Transcriptome Analysis Reveals Temperature-Dependent Agr Regulation and Suggests Overlaps with Other Regulons

Listeria monocytogenes is a ubiquitous, opportunistic pathogenic organism. Environmental adaptation requires constant regulation of gene expression. Among transcriptional regulators, AgrA is part of an auto-induction system. Temperature is an environmental cue critical for in vivo adaptation. In order to investigate how temperature may affect AgrA-dependent transcription, we compared the transcriptomes of the parental strain L. monocytogenes EGD-e and its Delta agrA mutant at the saprophytic temperature of 25 degrees C and in vivo temperature of 37 degrees C. Variations of transcriptome were higher at 37 degrees C than at 25 degrees C. Results suggested that AgrA may be involved in the regu…

Métabolisme sucre-citrate chez Leuconostoc mesenteroides

Sugar citrate cometabolism in Leuconostoc mesenteroides. Bacteria from the genus Leuconostocplay roles in the dairy industry. The most important functions of this bacteria are their ability to produce CO 2 and flavour compounds through lactose heterofermentation and citrate uti- lization. Although the biotechnological role of the citrate metabolism is very important and widely appreciated, little is known about the genetic properties of Leuconostoc spp. In our laboratory, we cloned the genes responsible for citrate metabolism ( clyR mae citCDEFGOPcluster), for D-lactate dehydrogenase (ldhD) and for phosphotransacetylase ( pta). In addition we have planned to con- struct new vectors and we h…

Changes in gene expression during adaptation of Listeria monocytogenes to the soil environment

project SEST 009; International audience; Listeria monocytogenes is a ubiquitous opportunistic pathogen responsible for listeriosis. In order to study the processes underlying its ability to adapt to the soil environment, whole-genome arrays were used to analyse transcriptome modifications 15 minutes, 30 minutes and 18 h after inoculation of L. monocytogenes EGD-e in soil extracts. Growth was observed within the first day of incubation and large numbers were still detected in soil extract and soil microcosms one year after the start of the experiment. Major transcriptional reprofiling was observed. Nutrient acquisition mechanisms (phosphoenolpyruvate-dependent phosphotransferase systems and…