Assembly of D-alanyl-lipoteichoic acid in Lactobacillus casei: mutants deficient in the D-alanyl ester content of this amphiphile

Abstract

D-Alanyl-lipoteichoic acid (D-alanyl-LTA) from Lactobacillus casei ATCC 7469 contains a poly(glycerophosphate) moiety that is acylated with D-alanyl ester residues. The physiological function of these residues is not well understood. Five mutant strains of this organism that are deficient in the esters of this amphiphile were isolated and characterized. When (15,20). One of the components of LTA which has been proposed to modulate these functions is the substituent D-alanine. Evidence from in vitro experiments indicates that these D-alanyl esters alter the abilities of LTA to inhibit autolysins (19), chelate Mg2+ ions (a function shared with wall teichoic acid) (2,21,24,28), and function as a carrier for wall teichoic acid synthesis (17, 27). Fischer and co-workers (19) proposed that fluctuations of the D-alanyl ester content of LTA must be considered in its potential regulation of autolysins.Substitution of LTA by D-alanine is not a universal feature of all LTAs. For example, LTAs from Micrococcus varians, Streptococcus faecalis 9790, and S. lactis Kiel 42172 are devoid of D-alanine (16). However, 30 to 93% of the glycerol phosphate units of LTAs from most organisms are substituted with D-alanyl ester residues (16). Growth of Staphylococcus aureus in the presence of increasing concentrations of NaCl, 0.2% to 7.5%, decreases the degree of esterification by D-alanine from 78 to 36% (18). In addition, growth of this organism at pH 8.1 instead of pH 7.1 results in a decrease of D-alanine ester content from 45 to 5% (32). Thus, not only growth in NaCl but growth at higher pHs induces wide variations in the esterification of LTA by D-alanine.In Lactobacillus casei, incorporation of D-alanine into LTA is accomplished in the following two-step reaction sequence (4,7,10,30,36,39 It is our ultimate goal to determine the physiological function of the D-alanyl ester residues in the LTA of L. casei 7469. In this paper, we report the isolation and characterization of five mutant strains of this organism that are deficient in the ester content of this amphiphile. A study of these strains revealed defects in the D-alanine incorporation system. Aberrant morphology and defective cell separation appear to result from this deficiency in D-alanyl ester content.(A portion of this study was presented at the 85th Annual Meeting of the American Society for Microbiology, Las Vegas, Nev., 3-7 March, 1985.) MATERIALS AND METHODS Materials. We thank Werner Fischer for glycerophospho (Gro-P)-6-Glc(pl-6)Gal(al-2)Glc(al-3)diacylglycerol and lipid extract isolated from L. casei DSM 20021 (34, 35).

Biosynthesis of D-alanyl-lipoteichoic acid: cloning, nucleotide sequence, and expression of the Lactobacillus casei gene for the D-alanine-activating enzyme

“…Many of these homologous enzymes are also known to catalyze nonribosomal peptide condensation via a thiolester intermediate (32) One of the proposed functions of LTA and wall teichoic acid in gram-positive organisms is metal ion chelation, specifically the binding of Mg2e (5,30 (52). Fischer et al (22) proposed that the D-alanine ester content of LTA may function in the regulation of autolysins.…”

“…Mutants that are deficient in these esters are characterized by aberrant morphology and defective cell separation (52). To establish further the role of these esters, genetic methods were sought for modulating their content in LTA.…”

D-Alanyl-lipoteichoic acid in Lactobacillus casei: secretion of vesicles in response to benzylpenicillin

“…4~~-benzylpenicillin, vesicle formation must occur by a mechanism different from that in cellular lysis. The MIC of this B-lactam is 2.2 PM (Ntamere et al, 1987). In Fig.…”

“…The D-alanine esters of LTA may provide one of the mechanisms for modulating these functions. To study the pathway for the D-alanylation of LTA in Lactobacillus casei 7469 (Ntamere et al, 1987), it was proposed that vesicles secreted in the presence of penicillin might provide an in vitro acceptor system that would conserve the in vivo organization of the LTA and associated proteins.…”

Characterization of Two Component Systems of Lactobacillus casei BL23 and their involvement in stress response

“…The inactivation of dltD in L. rhamnosus GG (ATCC 53103) resulted in 2.4-fold-increased cell length, a low survival capacity in response to gastric juice challenge, an increased sensitivity to human beta-defensin-2, an increased rate of autolysis, an increased capacity to initiate growth in the presence of an anionic detergent, and a decreased capacity to initiate growth in the presence of CAMPs compared to the wild-type (Perea . L. casei mutants defective in LTA D-alanyl esters show aberrant C-shaped morphologies and defects in cell separation indicative of reduced autolytic activity (Ntamere et al, 1987;. On the contrary, dltA mutation in Lactobacillus reuteri 100-23 did not have an effect on cell morphology and growth characteristics (Walter et al, 2007).…”