Dataset Information

Modeled Structure of the Cell Envelope Proteinase of Lactococcus lactis.

ABSTRACT: The cell envelope proteinase (CEP) of Lactococcus lactis is a large extracellular protease covalently linked to the peptidoglycan of the cell wall. Strains of L. lactis are typically auxotrophic for several amino acids and in order to grow to high cell densities in milk they need an extracellular protease. The structure of the entire CEP enzyme is difficult to determine experimentally due to the large size and due to the attachment to the cell surface. We here describe the use of a combination of structure prediction tools to create a structural model for the entire CEP enzyme of Lactococcus lactis. The model has implications for how the bacterium interacts with casein micelles during growth in milk, and it has implications regarding the energetics of the proteolytic system. Our model for the CEP indicates that the catalytic triad is activated through a structural change caused by interaction with the substrate. The CEP of L. lactis might become a useful model for the mode of action for enzymes belonging to the large class of S8 proteinases with a PA (protease associated) domain and a downstream fibronectin like domain.

SUBMITTER: Hansen EB

PROVIDER: S-EPMC7783315 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Modeled Structure of the Cell Envelope Proteinase of Lactococcus lactis.

Hansen Egon Bech EB Marcatili Paolo P

Frontiers in bioengineering and biotechnology 20201222

The cell envelope proteinase (CEP) of Lactococcus lactis is a large extracellular protease covalently linked to the peptidoglycan of the cell wall. Strains of L. lactis are typically auxotrophic for several amino acids and in order to grow to high cell densities in milk they need an extracellular protease. The structure of the entire CEP enzyme is difficult to determine experimentally due to the large size and due to the attachment to the cell surface. We here describe the use of a ...[more]

PMID: 33415101

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Project description:Lactococcus lactis is widely used as a starter in the manufacture of cheese and fermented milk. Its main role is the production of lactic acid, but also contributes to the sensory attributes of cheese. Unfortunately, the diversity of suitable strains to be commercialized as dairy starters is limited. In this work, we have applied adaptive evolution under cell envelope stress (AE-CES) as means to provide evolved L. lactis strains with distinct physiological and metabolic traits. A total of seven strains, three of industrial origin and four wild nisin Z-producing L. lactis, were exposed to subinhibitory concentrations of Lcn972, a bacteriocin that triggers the cell envelope stress response in L. lactis. Stable Lcn972 resistant (Lcn972R) mutants were obtained from all of them and two mutants per strain were further characterized. Minimal inhibitory Lcn972 concentrations increased from 4- to 32-fold compared to their parental strains and the Lcn972R mutants retained similar growth parameters in broth. All the mutants acidified milk to a pH below 5.3 with the exception of one that lost the lactose plasmid during adaptation and was unable to grow in milk, and two others with slower acidification rates in milk. While in general phage susceptibility was unaltered, six mutants derived from three nisin Z producers became more sensitive to phage attack. Loss of a putative plasmid-encoded anti-phage mechanism appeared to be the reason for phage susceptibility. Otherwise, nisin production in milk was not compromised. Different inter- and intra-strain-dependent phenotypes were observed encompassing changes in cell surface hydrophobicity and in their autolytic profile with Lcn972R mutants being, generally, less autolytic. Resistance to other antimicrobials revealed cross-protection mainly to cell wall-active antimicrobials such as lysozyme, bacitracin, and vancomycin. Finally, distinct and shared non-synonymous mutations were detected in the draft genome of the Lcn972R mutants. Depending on the parental strain, mutations were found in genes involved in stress response, detoxification modules, cell envelope biogenesis and/or nucleotide metabolism. As a whole, the results emphasize the different strategies by which each strain becomes resistant to Lcn972 and supports the feasibility of AE-CES as a novel platform to introduce diversity within industrial L. lactis dairy starters.

Project description:BACKGROUND: Membrane proteins comprise an important class of molecules whose study is largely frustrated by several intrinsic constraints, such as their hydrophobicity and added requirements for correct folding. Additionally, the complexity of the cellular mechanisms that are required to insert membrane proteins functionally in the membrane and to monitor their folding state makes it difficult to foresee the yields at which one can obtain them or to predict which would be the optimal production host for a given protein. METHODS AND FINDINGS: We describe a rational design approach to improve the lactic acid bacterium Lactococcus lactis as a producer of membrane proteins. Our transcriptome data shows that the two-component system CesSR, which senses cell envelope stresses of different origins, is one of the major players when L. lactis is forced to overproduce the endogenous membrane protein BcaP, a branched-chain amino acid permease. Growth of the BcaP-producing L. lactis strain and its capability to produce membrane proteins are severely hampered when the CesSR system itself or particular members of the CesSR regulon are knocked out, notably the genes ftsH, oxaA2, llmg_2163 and rmaB. Overexpressing cesSR reduced the growth defect, thus directly improving the production yield of BcaP. Applying this rationale to eukaryotic proteins, some of which are notoriously more difficult to produce, such as the medically-important presenilin complex, we were able to significantly diminish the growth defect seen in the wild-type strain and improve the production yield of the presenilin variant PS1?9-H6 more than 4-fold. CONCLUSIONS: The results shed light into a key, and perhaps central, membrane protein quality control mechanism in L. lactis. Modulating the expression of CesSR benefited the production yields of membrane proteins from different origins. These findings reinforce L. lactis as a legitimate alternative host for the production of membrane proteins.

Dataset Information

Modeled Structure of the Cell Envelope Proteinase of Lactococcus lactis.

Publications

Modeled Structure of the Cell Envelope Proteinase of <i>Lactococcus lactis</i>.

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