Project description:Bacteriophage infection of Lactococcus lactis strains used in the manufacture of fermented milk products is a major threat for the dairy industry. A greater understanding of the global molecular response of the bacterial host following phage infection has the potential to identify new targets for the design of phage control measures for biotechnological processes. In this study, we have used whole-genome oligonucleotide microarrays to gain insights into the genomic intelligence driving the instinctive response of L. lactis subsp. lactis IL1403 to the onset of a challenge with the lytic prolate-headed phage c2. Following phage adsorption, the bacterium differentially regulated the expression of 61 genes belonging to 14 functional categories, and mostly to cell envelope (12 genes), regulatory functions (11 genes), and carbohydrate metabolism (7 genes). The nature of the differentially regulated genes suggests the orchestration of a complex response involving induction of cell envelope stress proteins, D-alanylation of cell-wall lipoteichoic acids (LTAs), restoration of the proton motive force (PMF), and energy conservation. Increased D-alanylation of LTAs would act as an adsorption blocking mechanism, which we speculate may allow the survival of a small percent of the cell population when facing more realistic in vivo low titer-phage attacks. The modification of LTAs decoration in response to phage c2 adsorption also suggests these cell wall structures as possible primary receptors for this phage. Restoration of a physiological PMF is achieved by regulating the expression of genes affecting the two main components of the PMF, and serves to reverse a drastic depolarization of the host membrane caused by phage adsorption. Down-regulation of energy-consuming metabolic activities and a switch to anaerobic respiration helps the bacterium to save energy in order to sustain the PMF and the overall response to phage. We finally propose that the overall transcriptional response of L. lactis IL1403 to the phage stimuli is orchestrated by the concerted action of Phage Shock Proteins and of the bivalent transcriptional regulator SpxB following activation by the two-component system CesSR. To our knowledge, this represents the first detailed description in L. lactis, and probably in Gram-positive bacteria, of the molecular mechanisms involved in the host response to the membrane perturbation mediated by phage adsorption. Two-condition experiment: IL1403 vs. Bacteriophage c2-infected IL1403 cells. Biological replicates: 2 controls, 2 infected, independently grown and harvested. Two technical replicates per array.

Project description:Bacteriophage infection of Lactococcus lactis strains used in the manufacture of fermented milk products is a major threat for the dairy industry. A greater understanding of the global molecular response of the bacterial host following phage infection has the potential to identify new targets for the design of phage control measures for biotechnological processes. In this study, we have used whole-genome oligonucleotide microarrays to gain insights into the genomic intelligence driving the instinctive response of L. lactis subsp. lactis IL1403 to the onset of a challenge with the lytic prolate-headed phage c2. Following phage adsorption, the bacterium differentially regulated the expression of 61 genes belonging to 14 functional categories, and mostly to cell envelope (12 genes), regulatory functions (11 genes), and carbohydrate metabolism (7 genes). The nature of the differentially regulated genes suggests the orchestration of a complex response involving induction of cell envelope stress proteins, D-alanylation of cell-wall lipoteichoic acids (LTAs), restoration of the proton motive force (PMF), and energy conservation. Increased D-alanylation of LTAs would act as an adsorption blocking mechanism, which we speculate may allow the survival of a small percent of the cell population when facing more realistic in vivo low titer-phage attacks. The modification of LTAs decoration in response to phage c2 adsorption also suggests these cell wall structures as possible primary receptors for this phage. Restoration of a physiological PMF is achieved by regulating the expression of genes affecting the two main components of the PMF, and serves to reverse a drastic depolarization of the host membrane caused by phage adsorption. Down-regulation of energy-consuming metabolic activities and a switch to anaerobic respiration helps the bacterium to save energy in order to sustain the PMF and the overall response to phage. We finally propose that the overall transcriptional response of L. lactis IL1403 to the phage stimuli is orchestrated by the concerted action of Phage Shock Proteins and of the bivalent transcriptional regulator SpxB following activation by the two-component system CesSR. To our knowledge, this represents the first detailed description in L. lactis, and probably in Gram-positive bacteria, of the molecular mechanisms involved in the host response to the membrane perturbation mediated by phage adsorption.

Project description:Recently, we demonstrated that fermentation conditions strongly impact on subsequent survival of Lactococcus lactis strain MG1363 during heat and oxidative stress, two important parameters during spray drying. Moreover, employment of a transcriptome-phenotype matching approach revealed groups of genes associated with robustness towards heat and/or oxidative stress. To investigate if other strains have similar or distinct transcriptome signatures for robustness, we applied a similar transcriptome-robustness phenotype matching approach on the L. lactis strains IL1403, KF147 and SK11. These strains were employed in an identical fermentation regime as was performed earlier for strain MG1363 which consisted of twelve conditions, varying in the presence of salt and/or oxygen, as well as fermentation temperature and pH. In the exponential phase of growth, cells were harvested for transcriptome analysis and assessment of heat and oxidative stress survival phenotypes. The variation in fermentation conditions resulted in differences in heat and oxidative stress survival up to five 10-log units. Effects of the fermentation conditions on stress survival of the L. lactis strains were typically strain-dependent, although the fermentation conditions had mainly similar effects on the growth characteristics of the different strains. Association of transcriptomes and robustness phenotypes identified highly strain-specific transcriptome signatures for robustness, indicating that multiple mechanism exist to increase robustness and, as a consequence, robustness of each strain requires individual optimization. However, a relatively small overlap in the transcriptome responses of the strains was also identified and this generic transcriptome signature included genes previously associated with stress (ctsR and lplL) and novel genes, including nanE and genes encoding transport proteins. The transcript levels of these genes can function as indicators of robustness and could aid in selection of fermentation parameters, potentially resulting in more optimal robustness during spray drying.

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Host intelligence in response to bacteriophage c2 infection in Lactococcus lactis IL1403

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