Proteomic expression of microfungal ripening starter Geotrichum candidum submitted to cold stress is strain-dependent: studies using 2d-dige technology and samespots software analysis.

Geotrichum candidum is a micro-fungus widely used as a ripening starter in cheese making. In anthropogenic environments such as dairy industries, this microorganism is subjected to many environmental and technological stresses including low temperature exposure. Our aim was to study the proteomic response of G. candidum to cold stress using a comparative proteomic approach by two-dimensional Differential In Gel Electrophoresis (2D DIGE). This technique consists on the labeling of proteins by specific fluorescent dyes (CyDyes). The results, obtained with G. candidum cells subjected to cold temperature, show significant proteomic patterns differences compared with the standard conditions. Furthermore, this biochemical response seems strain specific. 2D DIGE technology combined with SameSpots™ software analysis support these results through an important statistical validity. The comparative studies in a single gel, using two different fluorescent CyDyes (Cy3 and Cy5), lead to proteins differentiation. Selected spots were treated and analyzed by mass spectrometry.

Relationship between growth behaviour, micro and macroscopic morphologies and freezing sensitivity of the ripening starter Geotrichum candidum is strain specific and mostly related to the morphotypes: the arthrospores/hyphae parameter.

Microscopic conformation, growth behaviour and freezing sensitivity of seven strains of Geotrichum candidum, a ripening starter, were studied and compared according to their macroscopic morphotypes. It has been shown that the thallus forming units (TFU)×ml-1/OD600nm ratio as a function of time is an interesting parameter to follow G. candidum sporulation through the growth behaviour. Microscopic conformation, growth behaviour and freezing sensitivity are clearly strain specific and mostly related to their corresponding morphotypes "yeast", "mould" or "intermediate". The two "mould" strains that sporulate weakly (UCMA103, UCMA499) showed a low survival rate to freezing stress whereas the "yeast" strains expressed a significant resistance owing to the arthrospore abundance. Interestingly, one strain (UCMA96) which appeared on solid medium in accord with the "mould" morphotype respond similarly to freezing stress.

Response to environmental stress as a global phenomenon in biology: the example of microorganisms.

Any modification of the environment that leads to a physiological, genetic, or epigenetic adaptive response in microorganisms may be considered as a stress. Historically, forms of stresses affecting biological structures were classified either as non-thermal, such as osmotic, oxidative, or acid stress or as thermal stress, hot or cold. Currently, the classification in biology is as abiotic, including physical and chemical stress, or biotic. The aim of this mini-review is to show, through the example of microorganisms, that the response to stress can be considered, in biology, as a global phenomenon, which can be extended to anthropogenic pressure.

Cryotolerance of Lactobacillus delbrueckii subsp. bulgaricus CFL1 is modified by acquisition of antibiotic resistance.

This study aimed to relate the acquisition of different antibiotic resistances and the corresponding physiological responses to cold stress of Lactobacillus delbrueckii subsp. bulgaricus strain CFL1. Six resistant mutants were spontaneously obtained and studied depending on the target of the antibiotic: (i) bacitracin and vancomycin (Bac(R), Van(R), wall synthesis), (ii) novobiocin (Nov(R), DNA replication), and (iii) kanamycin, spiramycin, streptomycin (Kan(R), Spi(R), Str(R), RNA translation). The mutations modified the growth and the cold stress response at three different physiological levels: (i) Van(R) and Spi(R) mutants showed significant lower growth rates compared to the wild type strain. (ii) Van(R) and Bac(R) mutants displayed a slightly higher resistance to a freezing-thawing challenge whereas Str(R) and Spi(R) mutants were more sensitive compared to the wild type. (iii) The recovery of acidification activity after freezing and during frozen storage was improved by considering the Nov(R) strain, but not with the Van(R) and Spi(R) mutants. Thus, acquisition of some antibiotic resistance by spontaneous mutation led to modification of the cold stress response. The hypothesis of a unique cellular thermostat is discussed regarding the diversity of the tested antibiotics.

Resin straw as an alternative system to securely store frozen microorganisms.

Freezing of prokaryotic and eukaryotic microorganisms is the main interest in the study of cold stress responses of living organisms. In parallel, applications which arise from this approach are of two types: (i) optimization of the frozen starters used in food processing; and (ii) improvement of the ex situ preservation of microorganisms in collections. Currently, cryopreservation of microorganisms in collections is carried out in cryotubes, and bibliographical references related to freezing microorganisms packaged in straws are scarce. In this context, a preliminary study was completed to evaluate the technological potential of ionomeric resin straws compared to polycarbonate cryo-tubes. Survival under freezing stress was tested on three microorganisms selected for their biotechnological interest: two lactic acid bacteria, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus and a deuteromycete fungus, Geotrichum candidum. The stress was carried out by repeated freezing-thawing cycles to artificially accelerate the lethal effect of freezing on the microorganisms. Two main results were obtained: (i) the survival rate values (per freezing-thawing cycle) seems to depend on the thermal type of the studied microorganism, and (ii) there was no, under our experimental conditions, significant difference between straws and tubes. However, conservation in the resin straws lead to a slight increase in the survival of L. cremoris and G. candidum compared to microtubes. In those conditions, straws seems an alternative system to securely store frozen microorganisms with three main characteristics: (i) a high resistance to thermal stress, (ii) a safe closing by hermetic weld, and (iii) a system for inviolable identification.

Nystatin and osmotica as chemical enhancers of the phenotypic adaptation to freeze-thaw stress in Geotrichum candidum ATCC 204307.

Geotrichum candidum is a yeast-like fungus used as ripening starter in cheese making. The present study focused on chemical stress pretreatments affecting survival of G. candidum ATCC 204307 to freeze-thaw stress. Cryotolerance of G. candidum cells was induced by pretreatment with NaCl, CaCl2, or MgCl2, indicating heterologous phenotypic adaptation to freeze-thaw stress (- 20 to 25 degrees C) by osmotic stress. Furthermore, the nystatin, an antifungal compound, was shown to be a cryotolerance inducer.