Lysis of a Lactococcus lactis Dipeptidase Mutant and Rescue by Mutation in the Pleiotropic Regulator CodY

Abstract

Lactococcus lactis subsp. cremoris MG1363 is a model for the lactic acid bacteria (LAB) used in the dairy industry. The proteolytic system, consisting of a proteinase, several peptide and amino acid uptake systems, and a host of intracellular peptidases, plays a vital role in nitrogen metabolism and is of eminent importance for flavor formation in dairy products. The dipeptidase PepV functions in the last stages of proteolysis. A link between nitrogen metabolism and peptidoglycan (PG) biosynthesis was underlined by the finding that deletion of the dipeptidase gene pepV (creating strain MGΔpepV) resulted in a prolonged lag phase when the mutant strain was grown with a high concentration of glycine. In addition, most MGΔpepV cells lyse and have serious defects in their shape. This phenotype is due to a shortage of alanine, since adding alanine can rescue the growth and shape defects. Strain MGΔpepV is more resistant to vancomycin, an antibiotic targeting peptidoglycan d-Ala–d-Ala ends, which confirmed that MGΔpepV has an abnormal PG composition. A mutant of MGΔpepV was obtained in which growth inhibition and cell shape defects were alleviated. Genome sequencing showed that this mutant has a single point mutation in the codY gene, resulting in an arginine residue at position 218 in the DNA-binding motif of CodY being replaced by a cysteine residue. Thus, this strain was named MGΔpepVcodYR218C. Transcriptome sequencing (RNA-seq) data revealed a dramatic derepression in peptide uptake and amino acid utilization in MGΔpepVcodYR218C. A model of the connections among PepV activity, CodY regulation, and PG synthesis of L. lactis is proposed. IMPORTANCE Precise control of peptidoglycan synthesis is essential in Gram-positive bacteria for maintaining cell shape and integrity as well as resisting stresses. Although neither the dipeptidase PepV nor alanine is essential for L. lactis MG1363, adequate availability of either ensures proper cell wall synthesis. We broaden the knowledge about the dipeptidase PepV, which acts as a linker between nitrogen metabolism and cell wall synthesis in L. lactis.

Invited review: Starter lactic acid bacteria survival in cheese: New perspectives on cheese microbiology

“…Further evidence was recently provided by Huang et al (2020) for a relationship between proteolysis, cell wall synthesis pathways and resultant autolytic phenotype of Lc. lactis subsp.…”

“…Following the detection of a new subpopulation of starter cells by Yanachkina et al (2020), we can propose an overall view of LAB viability (Figure 2) showing strain-related differences in accessibility to PepX substrates following cooking temperature, but which persist during storage in cheese model systems. It is possible that this subpopulation has particular phenotypic advantages and may form the nucleus of the extended survivor cell subpopulation akin to the MGΔPepV* cells noted by Huang et al (2020). Studies have indicated that permeabilization of strains such as L. lactis subsp.…”

Editing of the Proteolytic System of Lactococcus lactis Increases Its Bioactive Potential

“…When peptidases with similar specificities are removed, such as in strain MGΔ pepXPQ , lacking several proline-specific peptidases, or in a strain deficient for peptidases playing important roles in the last stages of peptide degradation (the di-/tripeptidase mutant MGΔ pepVD A TD B ), a dramatic decrease in the number of unique identified peptides is seen relative to that in strain MG1363. Strain MGΔ pepVD A TD B produced less than half the PSMs of MG1363, which might be due to the fact that deletion of pepV affects cell wall synthesis, which ultimately disturbs other biological processes such as nitrogen metabolism ( 31 ).…”

“…No bioactive peptides were observed when using MGΔ pepVD A TD B to degrade β-casein, and only 2 were found when employing strain MGΔ pepXPQ . As explained above, these might be caused by the disruption of other biological processes, such as peptidoglycan biosynthesis, when deleting the dipeptidase PepV ( 31 ).…”

“…When a group of specialty peptidases is removed, a problem might arise if they not only are responsible for β-casein digestion but also are important in another metabolic pathway(s), disrupting certain essential processes. A clear example is the role that PepV plays in peptidoglycan synthesis ( 31 ).…”