Enzymatic activities and functional interdependencies of Bacillus subtilis lipoteichoic acid synthesis enzymes.

Mirka E Wörmann,Peter J Simpson,Angelika Gründling,Rebecca M Corrigan,Steve J Matthews

doi:10.1111/j.1365-2958.2010.07472.x

Mirka E Wörmann, Peter J Simpson + Show 3 more

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https://doi.org/10.1111/j.1365-2958.2010.07472.x

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Abstract

Lipoteichoic acid (LTA) is an important cell wall polymer in Gram-positive bacteria. The enzyme responsible for polyglycerolphosphate LTA synthesis is LtaS, first described in Staphylococcus aureus. Four LtaS orthologues, LtaSBS, YfnI, YqgS and YvgJ, are present in Bacillus subtilis. Using an in vitro enzyme assay, we determined that all four proteins are Mn2+-dependent metal enzymes that use phosphatidylglycerol as a substrate. We show that LtaSBS, YfnI and YqgS can produce polymers, suggesting that these three proteins are bona-fide LTA synthases while YvgJ functions as an LTA primase, as indicated by the accumulation of a GroP-Glc2-DAG glycolipid. Western blot analysis of LTA produced by ltaSBS, yfnI, yqgS and yvgJ single, triple and the quadruple mutant, showed that LTA production was only abolished in the quadruple and the YvgJ-only expressing mutant. B. subtilis strains expressing YfnI in the absence of LtaSBS produced LTA of retarded mobility, presumably caused by an increase in chain length as suggested by a structural analysis of purified LTA. Taken together, the presented results indicate that the mere presence or absence of LTA cannot account for cell division and sporulation defects observed in the absence of individual enzymes and revealed an unexpected enzymatic interdependency of LtaS-type proteins in B. subtilis.

Highlights

Teichoic acids are important cell wall components in Gram-positive bacteria
Using an in vitro enzyme assay system, we have recently shown that the purified S. aureus eLtaS domain is sufficient to cleave the head group of fluorescently labelled PG producing diacylglycerol (DAG) and presumably glycerolphosphate, providing further evidence that this lipid is the physiological substrate for LtaS and Lipoteichoic acid (LTA) synthesis (Karatsa-Dodgson et al, 2010)
It was determined that the purified enzymatic domain of S. aureus LtaS, eLtaS, but not the active site variant eLtaS-T300A hydrolyses the glycerolphosphate head group of the fluorescently labelled lipid NBD-labelled PG (NBD-PG) resulting in the production of fluorescently labelled diacylglycerol (NBD-DAG) (Fig. 1A)

Summary

Introduction

Teichoic acids are important cell wall components in Gram-positive bacteria. Usually, two types of teichoicThe enzyme responsible for polyglycerolphosphate LTA backbone synthesis is LtaS, first described in S. aureus (Gründling and Schneewind, 2007a). The full-length protein is cleaved during growth and the eLtaS domain is released into the culture supernatant as well as partially retained within the cell wall fraction (Lu et al, 2009). Pulse-chase experiments have provided strong experimental evidence that the glycerolphosphate subunits of the LTA backbone are derived from the membrane lipid phosphatidylglycerol (PG) (Emdur and Chiu, 1974; 1975; Koch et al, 1984). Using an in vitro enzyme assay system, we have recently shown that the purified S. aureus eLtaS domain is sufficient to cleave the head group of fluorescently labelled PG producing diacylglycerol (DAG) and presumably glycerolphosphate, providing further evidence that this lipid is the physiological substrate for LtaS and LTA synthesis (Karatsa-Dodgson et al, 2010)

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