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).

Vesicles containing lipoteichoic acid (LTA) have been isolated from Lactobacillus casei ATCC 7469 grown in the presence of either benzylpeniciUin or D-cycloserine. These cell wall antibiotics enhanced the rate of LTA and lipid secretion 6.7 times, whereas chloramphenicol inhibited their release. The formation of these vesicles from peripheral and septal wall regions did not appear to be the result of bacteriolysis. The vesicle composition of LTA and lipid was similar to that of the cytoplasmic membrane whereas the protein composition was dissimilar. The size of these vesicles ranged from 20 to 40 nm and the length of LTA ranged from 5 to 50 glycerol phosphate residues. The isolation of these vesicles provides a potential in vitro acceptor system for studying the D-alanylation of lipoteichoic acid.

We have developed an in vitro translation system for the lower eukaryote Dictyostelium discoideum. Active extracts using endogenous mRNA support protein synthesis with optimal Mg2+ and K+ concentrations of 5 mM and 120 mM, respectively. [35S]Methionine incorporation is linear for more than 2 h. Polypeptides synthesized from endogenous mRNA have sizes ranging from less than 20 to over 100 kDa. Heat‐shock proteins are synthesized in vitro in extracts prepared from heat‐shocked cells. Possible uses of this system for study of translational control during growth and differentiation are discussed.