Universal Stress Proteins Are Important for Oxidative and Acid Stress Resistance and Growth of Listeria monocytogenes EGD-e In Vitro and In Vivo

Background: Pathogenic bacteria maintain a multifaceted apparatus to resist damage caused by external stimuli. As part of this, the universal stress protein A (UspA) and its homologues, initially discovered in Escherichia coli K-12 were shown to possess an important role in stress resistance and growth in several bacterial species. Methods and Findings: We conducted a study to assess the role of three homologous proteins containing the UspA domain in the facultative intracellular human pathogen Listeria monocytogenes under different stress conditions. The growth properties of three UspA deletion mutants (deltalmo0515, deltalmo1580 and deltalmo2673) were examined either following challenge with a sublethal concentration of hydrogen peroxide or under acidic conditions. We also examined their ability for intracellular survival within murine macrophages. Virulence and growth of usp mutants were further characterized in invertebrate and vertebrate infection models. Tolerance to acidic stress was clearly reduced in Δlmo1580 and deltalmo0515, while oxidative stress dramatically diminished growth in all mutants. Survival within macrophages was significantly decreased in deltalmo1580 and deltalmo2673 as compared to the wild-type strain. Viability of infected Galleria mellonella larvae was markedly higher when injected with deltalmo1580 or deltalmo2673 as compared to wild-type strain inoculation, indicating impaired virulence of bacteria lacking these usp genes. Finally, we observed severely restricted growth of all chromosomal deletion mutants in mice livers and spleens as compared to the load of wild-type bacteria following infection. Conclusion: This work provides distinct evidence that universal stress proteins are strongly involved in listerial stress response and survival under both in vitro and in vivo growth conditions

Lactobacillus pentosus genes involved in olive brine stress response

Background: Olives can be consumed only after a fermentation process driven by lactic acid bacteria (LAB) and yeasts. Only few adapted strains of LAB are able to grow and ferment table olives. In fact olive brine represents a stressful environment due to high NaCl concentration, presence of phenolic compounds known as antimicrobials, and low availability of nutrients. Objectives and methods: Lactobacillus pentosus C11 is a particularly resistant strain isolated from naturally fermented table olives. In order to identify the mechanisms by which this strain is able to tolerate olive brine stress conditions, a random transposition mutagenesis using the Pjunc-TpaseIS1223-based system was carried out. A library of 6000 mutants was generated and screened for the ability to grow in a brine based medium. Results and conclusion: Five transposition mutants impaired in growth on brine based medium were identified. The genotypic analysis allowed the identification of 5 genes involved in brine stress response of L. pentosus C11

Identification of Critical Genes for Growth in Olive Brine byTransposon Mutagenesis of Lactobacillus pentosus C11

Olive brine represents a stressful environment due to the high NaCl concentration, presence of phenolic compounds known as antimicrobials, and low availability of nutrients. Thus, only a few strains of lactic acid bacteria (LAB) are adapted to grow in and ferment table olives. To identify the mechanisms by which these few strains are able to grow in olive brine, Lactobacillus pentosus C11, a particularly resistant strain isolated from naturally fermented table olives, was mutagenized by random transposition using the Pjunc-TpaseIS1223 system (H. Licandro-Seraut, S. Brinster, M. van de Guchte, H. Scornec, E. Maguin, P. Sansonetti, J. F. Cavin, and P. Serror, Appl. Environ. Microbiol. 78:5417–5423, 2012). A library of 6,000 mutants was generated and screened for adaptation and subsequent growth in a medium, named BSM (brine screening medium), which presents the stressful conditions encountered in olive brine. Five transposition mutants impaired in growth on BSM were identified. Transposition occurred in two open reading frames and in three transcription terminators affecting stability of transcripts. Thus, several essential genes for adaptation and growth of L. pentosus C11 in olive brine were identified

Lactic acid bacteria in table olives: from identification to mutagenesis

Table olives are a very important component of the Mediterranean diet, but their production is still craft-based. The fermentation process depends on the indigenous microbiota, and it is often not standardized and difficult to be controlled. For this reason there is an increasing interest for starter culture development for olive fermentation, especially for lactic acid bacteria (LAB) since they play an important role for the safety and quality of the product.On the basis of the above considerations, our research was aimed at investigating the bacterial biodiversity table olives from different Italian cultivars. In particular, LAB were identified by a phenotypic and genetic approach based on 16S rRNA and recA gene sequencing and strains were typed by rep-PCR. To establish the molecular mechanisms that allow strain surviving in a stressful environment such as brine, a transposon mutagenesis was developed. In order to establish the randomness of the mutagenesis a Southern blot analysis was performed, which showed that the mutagenesis was random underling the efficiency of the developed strategy. A library of 6000 random transposon-mutants was generated. The development of an adapted screening strategy is now necessary to detect genes involved in the adaptation of Lactobacillus pentosus RT1 in brine. The obtained results should be useful for the selection of LAB to be used as starter cultures.[...

Identification of Critical Genes for Growth in Olive Brine by Transposon Mutagenesis of Lactobacillus pentosus C11

Olive brine represents a stressful environment due to the high NaCl concentration, presence of phenolic compounds known as antimicrobials, and low availability of nutrients. Thus, only a few strains of lactic acid bacteria (LAB) are adapted to grow in and ferment table olives. To identify the mechanisms by which these few strains are able to grow in olive brine, Lactobacillus pentosus C11, a particularly resistant strain isolated from naturally fermented table olives, was mutagenized by random transposition using the P-junc-TpaseIS1223 system (H. Licandro-Seraut, S. Brinster, M. van de Guchte, H. Scornec, E. Maguin, P. Sansonetti, J. F. Cavin, and P. Serror, Appl. Environ. Microbiol. 78:5417-5423, 2012). A library of 6,000 mutants was generated and screened for adaptation and subsequent growth in a medium, named BSM (brine screening medium), which presents the stressful conditions encountered in olive brine. Five transposition mutants impaired in growth on BSM were identified. Transposition occurred in two open reading frames and in three transcription terminators affecting stability of transcripts. Thus, several essential genes for adaptation and growth of L. pentosus C11 in olive brine were identified

In Lactobacillus pentosus, the olive brine adaptation genes are required for biofilm formation

International audienceLactobacillus pentosus is one of the few lactic acid bacteria (LAB) species capable of surviving in olive brine, and thus desirable during table olive fermentation. We have recently generated mutants of the efficient strain L. pentosus C11 by transposon mutagenesis and identified five mutants unable to survive and adapt to olive brine conditions. Since biofilm formation represents one of the main bacterial strategy to survive in stressful environments, in this study, the capacity of adhesion and formation of biofilm on olive skin was investigated for this strain and five derivative mutants which are interrupted in metabolic genes (enoA1 and gpi), and in genes of unknown function ("oba" genes). Confocal microscopy together with bacteria count revealed that the sessile state represented the prevailing L pentosus C11 life-style during table olive fermentation. The characterization of cell surface properties showed that mutants present less hydrophobic and basic properties than the wild type (WT). In fact, their ability to adhere to both abiotic (polystyrene plates) and biotic (olive skin) surfaces was lower than that of the WT. Confocal microscopy revealed that mutants adhered sparsely to the olive skin instead of building a thin, multilayer biofilm. Moreover, RT-qPCR showed that the three genes enoA1, gpi and obaC were upregulated in the olive biofilm compared to the planktonic state. Thus enoA1, gpi and "oba" genes are necessary in L pentosus to form an organized biofilm on the olive skin