Biosynthesis of the polysialic acid capsule in Escherichia coli K1. Cold inactivation of sialic acid synthase regulates capsule expression below 20°C*

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Abstract
When neuroinvasive Escherichia coli K1 cells are grown at temperatures below 20°C, they fail to synthesize the α-2,8linked polysialic acid (polySia) capsule. The objective of this study was to use a genetic and biochemical approach to analyse why capsule expression was defective at cold temperatures. The strategy was to construct E.coli K1-derived mutants with defects in activation and degradation of Sia. The inability to degrade Sia because of a defect in the Sia-specific aldolase permitted accurate quantitation of Sia and CMPSia. Strains EV5 and EV90 possessed a defective CMP-Sia synthetase and were unable to activate Sia. These mutants were then used to study how synthesis of Sia, CMP-Sia, and the polySia capsule was affected by growth at 15°C. In contrast to wild type strains, the mutants accumulated Sia in considerable quantities (up to 100 nmol mg protein−1) at 37°C. However, no Sia was detected after growth at 15°C. A temperature upshift experiment showed that the intracellular concentration of Sia increased ca. 3-fold within 5–10 min after shift from 15 to 37°C, even in the presence of inhibitors of protein synthesis or transcription initiation. An in vitro assay for Sia synthase showed that Sia was synthesized at 37°C in cell-free extracts from both 37 and 15°C grown cells, but that no synthesis occurred when the same extracts were assayed at 15°C. These results indicated that Sia synthase was a cold sensitive enzyme that was synthesized at 15°C, but was reversibly inactivated at low temperatures. Radiolabelling experiments using [14C]Sia showed that CMP-Sia synthetase and the polyST polymerase were also cold sensitive. We conclude that polySia capsule synthesis in E.coli K1 strains at 15°C is regulated primarily at the level of Sia synthase, rather than transcriptionally controlled.

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