Defects in D-alanyl-lipoteichoic acid synthesis in Streptococcus mutans results in acid sensitivity

In the cariogenic organism, Streptococcus mutans, low pH induces an acid to lerance response (ATR). To identify acid-regulated proteins comprising the ATR, transposon mutagenesis with the thermosensitive plasmid pGh9:ISS1 was used to produce clones that were able to grow at neutral pH, but not in med ium at pH 5.0. Sequence analysis of one mutant (IS1A) indicated that transp osition had created a 6.3-kb deletion, one end of which was in dltB of the dlt operon encoding four proteins (DltA-DltD) involved in the synthesis of D-alanyl-lipoteichoic acid. Inactivation of the dltC gene, encoding the D-a lanyl carrier protein (Dcp), resulted in the generation of the acid-sensiti ve mutant, BH97LC. Compared to the wild-type strain, LT11, the mutant exhib ited a threefold-longer doubling time and a 33% lower growth yield. In addi tion, it was unable to initiate growth below pH 6.5 and unadapted cells wer e unable to survive a 3-h exposure in medium buffered at pH 3.5, while a pH of 3.0 was required to kill the wild type in the same time period. Also, i nduction of the ATR in BH97LC, as measured by the number of survivors at a pH killing unadapted cells, was 3 to 4 orders of magnitude lower than that exhibited by the wild type. While the LTA of both strains contained a simil ar average number of glycerolphosphate residues, permeabilized cells of BH9 7LC did not incorporate D-[C-14]alanine into this amphiphile. This defect w as correlated with the deficiency of Dcp. Chemical analysis of the LTA puri fied from the mutant confirmed the absence of D-alanine-esters. Electron mi crographs showed that BH97LC is characterized by unequal polar caps and is devoid of a fibrous extracellular matrix present on the surface of the wild -type cells. Proton permeability assays revealed that the mutant was more p ermeable to protons than the wild type. This observation suggests a mechani sm for the loss of the characteristic acid tolerance response in S. mutans.