Dataset Information

Cryo-EM structure of GH31 alpha-1,3-glucosidase from Lactococcus lactis subsp. cremoris

ABSTRACT:

SUBMITTER: Naruhiko Adachi

PROVIDER: EMPIAR-11171 | biostudies-other |

REPOSITORIES: biostudies-other

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Publications

Structural basis of the strict specificity of a bacterial GH31 α-1,3-glucosidase for nigerooligosaccharides.

Ikegaya Marina M Moriya Toshio T Adachi Naruhiko N Kawasaki Masato M Park Enoch Y EY Miyazaki Takatsugu T

The Journal of biological chemistry 20220312 5

Carbohydrate-active enzymes are involved in the degradation, biosynthesis, and modification of carbohydrates and vary with the diversity of carbohydrates. The glycoside hydrolase (GH) family 31 is one of the most diverse families of carbohydrate-active enzymes, containing various enzymes that act on α-glycosides. However, the function of some GH31 groups remains unknown, as their enzymatic activity is difficult to estimate due to the low amino acid sequence similarity between characterized and u ...[more]

PMID: 35293315

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Project description:Lactococcus lactis is one of the most commonly used lactic acid bacteria in the dairy industry. Activation of competence for natural DNA transformation in this species would greatly improve the selection of novel strains with desired genetic traits. Here, we investigated the activation of natural transformation in L. lactis subsp. cremoris KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of proteins usually required for natural transformation. In the absence of knowledge about competence regulation in this species, we constitutively overproduced ComX in a reporter strain of late competence phase activation and showed, by transcriptomic analyses, a ComX-dependent induction of all key competence genes. We further demonstrated that natural DNA transformation is functional in this strain and requires the competence DNA uptake machinery. Since constitutive ComX overproduction is unstable, we alternatively expressed comX under the control of an endogenous xylose-inducible promoter. This regulated system was used to successfully inactivate the adaptor protein MecA and subunits of the Clp proteolytic complex, which were previously shown to be involved in ComX degradation in streptococci. In the presence of a small amount of ComX, the deletion of mecA, clpC, or clpP genes markedly increased the activation of the late competence phase and transformability. Altogether, our results report the functionality of natural DNA transformation in L. lactis and pave the way for the identification of signaling mechanisms that trigger the competence state in this species.IMPORTANCELactococcus lactis is a lactic acid bacterium of major importance, which is used as a starter species for milk fermentation, a host for heterologous protein production, and a delivery platform for therapeutic molecules. Here, we report the functionality of natural transformation in L. lactis subsp. cremoris KW2 by the overproduction of the master competence regulator ComX. The developed procedure enables a flexible approach to modify the chromosome with single point mutation, sequence insertion, or sequence replacement. These results represent an important step for the genetic engineering of L. lactis that will facilitate the design of strains optimized for industrial applications. This will also help to discover natural regulatory mechanisms controlling competence in the genus Lactococcus.

Dataset Information

Cryo-EM structure of GH31 alpha-1,3-glucosidase from Lactococcus lactis subsp. cremoris

Publications

Structural basis of the strict specificity of a bacterial GH31 α-1,3-glucosidase for nigerooligosaccharides.

Similar Datasets

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