Regulation of Cell Wall Plasticity by Nucleotide Metabolism in Lactococcus lactis

Abstract

To ensure optimal cell growth and separation and to adapt to environmental parameters, bacteria have to maintain a balance between cell wall (CW) rigidity and flexibility. This can be achieved by a concerted action of peptidoglycan (PG) hydrolases and PG-synthesizing/modifying enzymes. In a search for new regulatory mechanisms responsible for the maintenance of this equilibrium in Lactococcus lactis, we isolated mutants that are resistant to the PG hydrolase lysozyme. We found that 14% of the causative mutations were mapped in the guaA gene, the product of which is involved in purine metabolism. Genetic and transcriptional analyses combined with PG structure determination of the guaA mutant enabled us to reveal the pivotal role of the pyrB gene in the regulation of CW rigidity. Our results indicate that conversion of L-aspartate (L-Asp) to N-carbamoyl-L-aspartate by PyrB may reduce the amount of L-Asp available for PG synthesis and thus cause the appearance of Asp/Asn-less stem peptides in PG. Such stem peptides do not form PG cross-bridges, resulting in a decrease in PG cross-linking and, consequently, reduced PG thickness and rigidity. We hypothesize that the concurrent utilization of L-Asp for pyrimidine and PG synthesis may be part of the regulatory scheme, ensuring CW flexibility during exponential growth and rigidity in stationary phase. The fact that L-Asp availability is dependent on nucleotide metabolism, which is tightly regulated in accordance with the growth rate, provides L. lactis cells the means to ensure optimal CW plasticity without the need to control the expression of PG synthesis genes.Peptidoglycan (PG) 8 is the major component of the Grampositive bacterial cell wall (CW), which envelops the cell as a multilayer sacculus. PG consists of a basic unit made up of N-acetylglucosamine-N-acetylmuramic acid (GlcNAc-MurNAc) disaccharides bound to stem pentapeptides. Disaccharide pentapeptide units are synthesized intracellularly and transported through the cytoplasmic membrane as lipid-disaccharide pentapeptides called lipid II. These blocks are covalently linked to the pre-existing PG polymers by high molecular weight penicillin-binding proteins (PBPs) (1). Class A PBPs contain both transglycosylation and transpeptidation domains, whereas class B PBPs are involved only in transpeptidation. Transglycosylation links the disaccharide pentapeptide to the pre-existing PG chain, whereas transpeptidation connects the stem pentapeptides to neighboring chains, which ensures PG cross-linking through the formation of an interpeptide bridge. Cross-linking in Lactococcus lactis involves the synthesis of an interpeptide bridge made of one D-amino acid (D-Asp or D-Asn), and in this species the PG cross-linking index was estimated to be 35.5% (2). Studies of Staphylococcus aureus have revealed that this PG cross-linking correlates with CW rigidity (3).The basic PG structure is often modified as PG glycan chains can undergo N-deacetylation or O-acetylation, and free carboxyl groups of amino acids of peptide chains ...

Guanine Limitation Results in CodY-Dependent and -Independent Alteration of Staphylococcus aureus Physiology and Gene Expression

“…Then, as in B. subtilis, unphosphorylated RsbV would sequester RsbW, releasing SigB to interact with core RNA polymerase and stimulate transcription from SigB-dependent promoters. Alternatively, García-Betancur et al (77) recently showed that increased cell wall rigidity activates SigB, and connections between cell wall plasticity and nucleotide metabolism have also been reported (77,78). During our investigation, we found that GN limitation dysregulates a number of genes involved in cell wall and membrane phospholipid biosynthesis ( Fig.…”

Adaptive Evolution of Industrial Lactococcus lactis Under Cell Envelope Stress Provides Phenotypic Diversity

“…This mutation may have direct consequences on the architecture of the cell wall and turn cells resistant to cell wall antimicrobials as lysozyme and Lcn972. Recently, mutations in pyrB , coding for the aspartate carbamoyltransferase in pyrimidine metabolism, were frequently found in L. lactis resistance to lysozyme ( Solopova et al, 2016 ). The authors claimed that limiting the availability of L -Asp for nucleotide biosynthesis results in a highly cross-linked and rigid peptidoglycan that interferes with lysozyme activity.…”

Structural and functional studies of pyruvate carboxylase regulation by cyclic di-AMP in lactic acid bacteria

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