ABSTRACT
Lactobacillus (Lb.) sakei is widely used as starter in the production process of Italian fermented sausages and its growth and survival are affected by various factors such as temperature, pH and salt concentration. We studied the behaviour of Lb. sakei strains under various growth conditions relative to acid, osmotic and heat stress treatments by a novel fluorescent differential display (FDD) technique. This study obtained the development and the optimization of a technique that allows the identification of genome expression changes, associated with differential microbial behaviour under different stress conditions with a better stress response definition and a better discrimination of starter cultures. DNA sequence information from the FDD products provided an important tool to assess and observe the response to a variety of environmental stimuli and the adaptation to bacterial stress. Our work provided an innovative FDD method, with a high level of reproducibility and quality for studying and probing the knowledge of the relation between differential genome expression and different stresses tolerance.
Subject(s)
Gene Expression Profiling/methods , Lactobacillus/physiology , DNA Primers/genetics , Fluorescent Dyes/analysis , Gene Expression Regulation, Bacterial , Hot Temperature , Lactobacillus/genetics , Lactobacillus/growth & development , Stress, PhysiologicalABSTRACT
Oenococcus oeni strains from traditional Italian red wines of the Basilicata region were investigated on the basis of their physiological and molecular response to different temperatures and ethanol concentrations. All strains were highly resistant to different ethanol concentrations and it has been observed that 7% ethanol was able to stimulate the growth of strains in wine, and 12-13% of ethanol allowed their proliferation. Moreover, strain tolerances to 18 and 42 degrees C were observed. Fingerprinting analysis with fluorescent differential display-PCR and investigation of changes in gene expression during the tolerance process were carried out. The expression gene pattern reflects mechanisms involved in tolerance to environmental conditions. This study establishes and validates a method that enables, with a high reproducibility, different gene expression identification under stress conditions in lactic acid bacteria.
Subject(s)
Drug Resistance, Bacterial , Gene Expression Profiling , Gram-Positive Bacteria/physiology , Stress, Physiological , Anti-Bacterial Agents/pharmacology , Ethanol/pharmacology , Gram-Positive Bacteria/drug effects , Gram-Positive Bacteria/genetics , Gram-Positive Bacteria/radiation effects , Italy , Temperature , Wine/microbiologyABSTRACT
Lactobacillus sakei is widely used as starter in the production process of Italian fermented sausages and its growth and survival are affected by various factors. We studied the differential expression of genome in response to different stresses by the fluorescent differential display (FDD) technique. This study resulted in the development and optimization of an innovative technique, with a high level of reproducibility and quality, which allows the identification of gene expression changes associated with different microbial behaviours under different growth conditions.
Subject(s)
Gene Expression Regulation, Bacterial/physiology , Lactobacillus/physiology , Polymerase Chain Reaction/methods , Stress, Physiological , Fluorescence , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
The developmental protein Numb is a major determinant of binary cell fates. It is also required for the differentiation of cerebellar granule cell progenitors (GCPs) at a stage of development responsive to the morphogenic glycoprotein Hedehog. Hedgehog signalling is crucial for the physiological maintenance and self-renewal of neural stem cells and its deregulation is responsible for their progression towards tumorigenesis. The mechanisms that inhibit this pathway during the differentiation stage are poorly understood. Here, we identify Numb as a Hedgehog-pathway inhibitor that is downregulated in early GCPs and GCP-derived cancer cells. We demonstrate that the Hedgehog transcription factor Gli1 is targeted by Numb for Itch-dependent ubiquitination, which suppresses Hedgehog signals, thus arresting growth and promoting cell differentiation. This novel Numb-dependent regulatory loop may limit the extent and duration of Hedgehog signalling during neural-progenitor differentiation, and its subversion may be a relevant event in brain tumorigenesis.
